Analysis of trypsin digested IgG using capillary
electrophoresis and mass spectrometry
Ana Isabel Fernandes de Carvalho
Thesis to obtain the Master of Science Degree in
Chemical Engineering
Supervisors: Prof. Åsa Emmer;
Prof. Miguel Nobre Parreira Cacho Teixeira
Examination Committee
Chairperson: Prof. Maria Matilde Soares Duarte Marques
Supervisor: Prof. Miguel Nobre Parreira Cacho Teixeira
Members of the Committee: Prof. Ana Margarida Nunes da Mata Pires de Azevedo
November 2018
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Acknowledgments
I would first like to express my gratitude to Prof. Åsa Emmer for accepting me in her research group and
allowing me this great experience in KTH, for guiding me and for providing excellent conditions so that
my colleagues and I could have everything needed.
I wish to acknowledge Prof. Miguel Teixeira for his support, dedication, and interest during all this
process.
I am also grateful to all my colleagues of the Analytical Chemistry research group particularly to Sara
and Joakim for the friendship and guidance.
To António, thank you for your support and encouragement throughout all the process.
Finally, I would like to thank my family, specially my parents, for giving me this opportunity to study
abroad and for the great motivation along this journey.
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Abstract
Complex regional pain syndrome is a chronic condition, considered one of the most painful diseases.
Currently, there is no single diagnosis available which often leads to delaying the beginning of the
treatments. It could be hypothesized that mutations in the glycosylation of the protein Immunoglobin G,
the most abundant immunoglobulin in human plasma, could be a cause for this pathology.
This project includes the study of trypsin digested Immunoglobulin G. Capillary electrophoresis was
used for sample separation and mass spectrometry for identification. An evaluation of the systems and
optimization of the methods separately and in combination was carried out. Both electrospray ionization
mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry have been
investigated. Besides the implementation of the techniques mentioned, an effective method for coating
the capillaries with polyvinyl alcohol was developed.
Capillary electrophoresis revealed appropriate for the separation of Immunoglobulin G trypsin digested
peptides, with well-resolved electropherograms. With the electrospray ionization mass spectrometry, it
was not possible to identify IgG peptides neither performing a database search nor comparing with the
theoretical peptide mass list. Matrix-assisted laser desorption/ionization mass spectrometry proved to
be a powerful tool to identify the peptides. Numerous attempts were made to couple the separation
technique with mass spectrometry. However, it was not possible to develop a setup with reproducible
results.
Keywords: Capillary electrophoresis, mass spectrometry,
immunoglobulin G, complex regional pain syndrome
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Resumo
A Síndrome de Dor Regional Complexa é uma condição crónica, considerada uma das doenças que
provoca mais dor. Atualmente, não existe um diagnóstico disponível, o que leva muitas vezes ao atraso
do início do tratamento. Uma hipótese para a causa desta doença são mutações na glicosilação da
proteína Imunoglobulina G, a imunoglobulina mais abundante no plasma humano.
Este projeto inclui o estudo da imunoglobulina G, digerida através da enzima tripsina. A técnica de
eletroforese capilar foi utilizada para separação de amostra e a espectrometria de massas para
identificação desta proteína. Foi realizada uma avaliação dos sistemas e uma otimização dos métodos
de separação e identificação, tanto separadamente como acoplados. Ambas as técnicas de
espectrometria de massas de ionização por electrospray e de ionização/dessorção a laser assistida por
matriz foram investigadas. Além da implementação das técnicas mencionadas, foi ainda desenvolvido
um método para o revestimento de capilares com álcool polivinílico.
A eletroforese capilar revelou-se adequada para a separação dos péptidos da imunoglobulina G, tendo
sido obtidos eletroferogramas bem resolvidos. Utilizando a espectrometria de massas de ionização por
electrospray, não foi possível identificar os péptidos, nem através de pesquisa em bases de dados nem
comparando com a massa teórica. A espectrometria de massas de ionização/dessorção a laser
assistida por matriz mostrou-se uma ferramenta eficaz para identificar péptidos. Diversas tentativas
foram feitas para acoplar as técnicas de separação e espectrometria de massas, no entanto, não foi
possível desenvolver uma montagem experimental com resultados reprodutíveis.
Palavras-chave: Eletroforese capilar, espectrometria de massas,
imunoglobulina G, síndrome de dor regional complexa
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Table of Contents
Acknowledgments ................................................................................................................................ iii
Abstract .................................................................................................................................................. v
Resumo................................................................................................................................................. vii
Table of Contents ................................................................................................................................. ix
List of Figures ....................................................................................................................................... xi
List of Tables ....................................................................................................................................... xiii
List of Symbols ................................................................................................................................... xiii
List of Abbreviations .......................................................................................................................... xiv
1. Introduction .................................................................................................................................... 1
1.1. Context .................................................................................................................................. 1
1.2. Problem definition .................................................................................................................. 2
1.3. Objectives .............................................................................................................................. 2
1.4. Outline ................................................................................................................................... 3
2. Background .................................................................................................................................... 5
2.1. Fundamentals of Capillary Electrophoresis ........................................................................... 5
2.1.1. Capillary Wall Modifications ...................................................................................... 7
2.2. Fundamentals of Mass Spectrometry .................................................................................... 8
2.2.1. Electrospray Ionization ............................................................................................. 9
2.2.2. Matrix Assisted Laser Desorption/Ionization .......................................................... 10
2.3. Fundamentals of Immunoglobulin G .................................................................................... 11
2.4. Fundamentals of Proteomics ............................................................................................... 12
3. Materials and Methods ................................................................................................................ 15
3.1. Chemicals ............................................................................................................................ 15
3.2. Instrumentation .................................................................................................................... 15
3.2.1. Capillary Electrophoresis ........................................................................................ 16
3.2.2. Electrospray Ionization ........................................................................................... 17
3.2.3. Matrix Assisted Laser Desorption/Ionization .......................................................... 17
3.3. Sample Preparation ............................................................................................................. 18
3.4. Capillary Electrophoresis ..................................................................................................... 18
3.4.1. Capillary Wall Coating ............................................................................................ 19
3.5. Electrospray Ionization ........................................................................................................ 22
x
3.5.1. CE-ESI-MS Hyphenation ........................................................................................ 23
3.6. Matrix Assisted Laser Desorption/Ionization ....................................................................... 24
3.6.1. CE-MALDI-TOF-MS Hyphenation .......................................................................... 24
4. Results and Discussion .............................................................................................................. 29
4.1. Capillary Electrophoresis ..................................................................................................... 29
4.2. Electrospray Ionization ........................................................................................................ 34
4.2.1. CE-ESI-MS Hyphenation ........................................................................................ 39
4.3. Matrix Assisted Laser Desorption/Ionization ....................................................................... 40
CE-MALDI-TOF-MS Hyphenation ....................................................................................... 43
4.4. Comparison between ESI-MS and MALDI-TOF-MS ........................................................... 45
5. Conclusions and Future Works .................................................................................................. 47
6. Bibliography ................................................................................................................................. 49
A. Budapest Criteria for CRPS ........................................................................................................ 53
B. List of Digested Peptides from IgG ............................................................................................ 54
C. Biotools Analysis ......................................................................................................................... 69
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List of Figures
Figure 1 - Schematic representation of CE system ................................................................................. 5
Figure 2 – Schematic representation of tandem mass spectrometry ...................................................... 8
Figure 3 - Schematic representation of ESI-MS system (a) and of MALDI-MS (b)................................. 9
Figure 4 – Human IgG structure ............................................................................................................. 11
Figure 5 – Schematic representation of the mass-spectrometry/proteomic process ............................ 12
Figure 6 – Setup of the home-built CE instrument (voltage supply, main CE box, UV detector and
computer to collect the data, from left to right). ..................................................................................... 16
Figure 7 - Tool used for capillary wall coating ....................................................................................... 19
Figure 8 – CE electropherogram illustrating the impact of rinsing the capillary with NaOH ................. 20
Figure 9 – CE electropherogram of mesityl oxide 8,5 mg/mL; injection: 20 kV for 20 s; voltage: 25 kV;
buffer: sodium phosphate (pH=3.2; 100 mM) ........................................................................................ 22
Figure 10 – Apparatus used to verify the cut of the capillary ................................................................ 23
Figure 11 – Setup used for CE-ESI-MS analysis .................................................................................. 24
Figure 12 – CE-MALDI-MS setup ① .................................................................................................... 26
Figure 13 – CE-MALDI-MS setup ② .................................................................................................... 27
Figure 14 - CE electropherogram of digested bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s; voltage:
25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)............................................................................. 30
Figure 15 – CE electropherogram of intact bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s; voltage: 25
kV; buffer: sodium phosphate (pH=3.2; 100 mM) .................................................................................. 30
Figure 16 - CE electropherogram of digested bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s; voltage:
25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)............................................................................. 31
Figure 17 - CE electropherogram of digested bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s; voltage:
25 kV; buffer: sodium phosphate (pH=3.2; 50 mM) ............................................................................... 31
Figure 18 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s; voltage: 25
kV; buffer: sodium phosphate (pH=3.2; 100 mM) .................................................................................. 32
Figure 19 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s; voltage:
25 kV; buffer: ammonium acetate (pH=3.2; 100 mM) ........................................................................... 33
Figure 20 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s; voltage: 25
kV; buffer: ammonium acetate (pH=4.6; 100 mM) ................................................................................ 33
Figure 21 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s; voltage: 25
kV; buffer: ammonium acetate (pH=4.2; 25 mM) .................................................................................. 33
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Figure 22 - ESI-MS spectrum of digested human IgG 15 𝝁g/mL; scan mode: UltraScan; flow rate:
2 L/min; capillary voltage: 4 kV; dry gas flow: 6 L/min; dry gas temperature 300 °C; nebulizer pressure:
1 bar; mass tuning: 150 to 1500 m/z; target: 800 m/z ........................................................................... 34
Figure 23 – Zoomed in view of the peaks potentially related to IgG ..................................................... 36
Figure 24 - ESI-MS spectrum of digested human IgG 15 𝝁g/mL; scan mode: UltraScan; flow rate:
2 L/min; capillary voltage: 4.5 kV; drying gas flow: 6 L/min; drying gas temperature 300 °C; nebulizer
pressure: 0.9 bar; mass tuning: 300 to 1000 m/z; target: 500 m/z ........................................................ 37
Figure 25 – Zoomed in view of the peaks potentially related to IgG ..................................................... 37
Figure 26 – ESI-MS spectrum of digested human IgG 100 𝝁g/mL; scan mode: UltraScan; flow rate: 2
L/min; capillary voltage: 4 kV; drying gas flow: 4 L/min; drying gas temperature 300 °C; nebulizer
pressure: 0.9 bar; mass tuning: 200 to 1500 m/z; target: 800 m/z ........................................................ 38
Figure 27 – Zoomed in view of the peaks potentially related to IgG ..................................................... 39
Figure 28 - MALDI-TOF-MS spectrum of digested human IgG; matrix: DHB; shot at 90% laser intensity
(1000 shots per burst, 6000 shots total), pulsed ion extraction by reflectron ........................................ 40
Figure 29 - Results obtained from Biotools database search for MS analysis (database: Swiss-Prot) 41
Figure 30 - Results obtained from Biotools database search for tandem MS analysis (database: Swiss-
Prot) ....................................................................................................................................................... 42
Figure 31 – Results obtained from Biotools database search for tandem MS analysis of peak 1186.65
Da (database: Swiss-Prot)..................................................................................................................... 42
Figure 32 - CE electropherogram of digested human IgG 1 mg/mL; injection: 20 kV for 20 s; voltage: 25
kV; buffer: sodium phosphate (pH=3.2; 100 mM) .................................................................................. 43
Figure 33 - CE electropherogram of digested human IgG 1 mg/mL from CE-MALDI experimental setup
①; injection: 20 kV for 20 s; voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM) ................. 44
Figure 34 - CE electropherogram of digested human IgG 1 mg/mL from CE-MALDI experimental setup
②; injection: 20 kV for 20 s; voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM) ................. 45
Figure 35 – Results obtained from Biotools database search for MS analysis of IgG 1 ....................... 69
Figure 36 – Results obtained from Biotools database search for MS analysis of IgG 2 ....................... 69
Figure 37 – Results obtained from Biotools database search for MS analysis of IgG 3 ....................... 70
Figure 38 - Results obtained from Biotools database search for MS analysis of IgG 4 ........................ 70
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List of Tables
Table 1 – IgG sample list ....................................................................................................................... 18
Table 2 - Capillary parameters, wall coating procedure, symptoms and setup used ............................ 21
Table 3 – ESI peak list of digested human IgG 15 𝝁g/mL and comparison with theoretical data ......... 35
Table 4 – ESI peak list of digested human IgG 15 𝝁g/mL and comparison with theoretical data ......... 37
Table 5 – ESI peak list of digested human IgG 100 𝝁g/mL and comparison with theoretical data ....... 38
Table 6 - MALDI peak list of digested human IgG and comparison with theoretical data ..................... 41
Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 - 330 ................................. 54
Table 8 - Chain Immunoglobulin heavy constant gamma 2 at positions <1-326 ................................... 58
Table 9 - Immunoglobulin heavy constant gamma 3 at positions 1-377 ............................................... 62
Table 10 -– Immunoglobulin heavy constant gamma 4 at positions <1-327 ......................................... 66
List of Symbols
Symbol Meaning
𝐸 Applied Electric Field
𝑚/𝑧 Mass to Charge Ratio
𝑞 Charge
𝑟 Radius
𝑣 Velocity
𝜇𝐸𝑂𝐹 Electroosmotic Mobility
𝜇𝑒 Electrophoretic Mobility
𝜇 Viscosity
𝜁 Zeta Potential
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List of Abbreviations
Abbreviation Meaning
AKD
BGE
Alkyl Ketene Dimer
Background Electrolyte Solution
CE Capillary Electrophoresis
CRPS Complex Regional Pain Syndrome
CZE Capillary Zone Electrophoresis
DHB 2,5-Dihydroxybenzoic Acid
EOF Electroosmotic Flow
ESI Electrospray Ionization
HCCA α-cyano-4-hydroxycinnamic acid
IgG Immunoglobulin G
MALDI Matrix Assisted Laser Desorption/Ionization
MS Mass Spectrometry
MC Miss-cleavage
OPG Osteoprotegerin
PMF Peptide Mass Fingerprinting
PVA Polyvinyl Alcohol
RSD Reflex Sympathetic Dystrophy
SA 3,5-Dimethoxy-4-Hydroxycinnamic Acid
S/N Signal-to-noise
SL Sheath Liquid
TOF Time of Flight
UV Ultra-Violet
1
1. Introduction
1.1. Context
This project is part of a larger one that aims to develop instrumental bioanalytical setups and procedures
for the study of glycoproteins of potential importance in the pathophysiology and diagnosis of Complex
Regional Pain Syndrome (CRPS).
CRPS is a severe long-term pain syndrome, ranked as the most painful disease according to the McGill
Pain Index1 [1]. This condition shows a great variety of symptoms such as allodynia or hyperalgesia,
temperature and skin color asymmetry or trophic and motor changes. Nevertheless, chronic pain is the
key symptom, either permanent or fluctuating, and most often in the deep tissue; affecting one limb
(arm, leg, hand, or foot) [3, 4]. As the name suggests, this is a regional syndrome, i.e., not in a specific
nerve territory or dermatome [4].
There are two types of CPRS. CRPS type I, formerly described as Reflex Sympathetic Dystrophy (RSD),
is developed after any type physical of trauma, especially fractures, and soft tissue lesion. CRPS type II,
once referred to as causalgia, occurs after major nerve damage, which is not found in type I [4]. One
distinguishable characteristic is that CRPS type II does not migrate from the original site of injury like
CRPS type I.
The fact of CRPS being a multi-faceted pathophysiology makes the treatment more complex. There is
not one treatment that is effective for all cases. However, according to Dimova and Birklein (2017), a
systematic and interdisciplinary approach based on the following basic therapeutic principles is used
[2]:
(1) “Medical and nonmedical pain therapy (acute and chronic phases);
(2) Physiotherapy, occupational therapy and training therapy (acute and chronic phases);
(3) Anti-inflammatory therapy (acute phase);
(4) Psycho- and socio therapy in a multimodal treatment setting (especially targeting pain-related
fears; all phases if necessary);
(5) A limited number of sympathetic nerve blocks (in selected cases after successful test blocks;
(6) Therapy of dystonia.”
1 The McGill Pain Index is a scale that shows the rating level of pain, based on McGill Pain Questionnaire
(Melzack, 1975). The last is a three-part pain assessment tool evaluating the sensory intensity, the
emotional impact and the cognitive evaluation of pain.
2
Data from a population-based study estimates that this condition has an overall incidence rate of 26.2
per 100,000 person-years. The probability of being affected is at least three times higher in women than
in men [5].Though, the female preponderance might also be due to women suffering 3 times more radial
fractures [2]. The most affected group were women with ages between 61 and 70 years [5].
1.2. Problem definition
Currently, there is no single test to confirm this pathology. The diagnosis is made based on the patient´s
medical history and through the signs and symptoms presented that are in line with the definition of
CPRS. In the last decade, a new diagnostic criteria, the Budapest Criteria, established by the
International Association for the Study of Pain (IASP) has become accepted. [2]. More information is
presented in the appendix A. Budapest Criteria for CRPS.
However, the large range of symptoms and its similarity with many other diseases makes the diagnosis
highly challenging. [3]. Consequently, the diagnosis is made in most of the cases too late; even years
after the symptoms begin. Evidence suggests that the earlier specific treatment is started, the more
successful it is likely to be; by opposition a delay in the diagnosis and thus in the treatment can result in
severe secondary complications jeopardizing the quality of life for CRPS patients. Thus, improvement
of the diagnosis method is urgently needed.
1.3. Objectives
Discovering effective CRPS biomarkers to improve and quicken the diagnostic process has been under
the focus of the scientific community. Results from past research suggest that the pathophysiological
mechanism is multifactorial, with neuroinflammation, autoimmunity, and nociceptive sensitization among
the mechanisms thought to be involved; and that a single marker for CRPS is not likely to be found.
Thus, multiple biomarkers are needed to enhance the diagnosis. [8,9]. Several glycosylated proteins
are considered to play central roles in autoimmunity, inflammation and disturbed bone turnover
processes, and are thus of interest in relation to CRPS. Osteoprotegerin (OPG) has already been
reported as a biomarker, with elevated levels in the early phase of CRPS being indicative of acute bone
modifications. [8].
Immunoglobulin G (IgG) has been used as a biomarker in multiple diseases. That said, it is important to
clarify the concept of biomarkers. According to Kyle Strimbu and Jorge Tavel (2011), “biomarkers are
merely the most objective, quantifiable medical signs modern laboratory science allows us to measure
reproducibly”. Another definition by the World Health Organization (2001) refers to a biomarker as “any
substance, structure, or process that can be measured in the body or its products and influence or
predict the incidence of outcome or disease”. [11, 12].
3
Regarding CRPS, in a small pilot trial some patients with CRPS were given intravenous immunoglobulin
treatment. The results have demonstrated a significant reduction in pain symptoms when compared with
those given a placebo [11]. Another study, by Valéria Tékus et al. (2014), where a patient’s serum or IgG
fraction was injected into mice also describes the relevance of IgG. The study reports that features
resembling the human disease were shown in mice, supporting both hypothesis “that autoantibodies
may contribute to the pathophysiology of CRPS, and that autoantibody-removing therapies may be
effective treatments for longstanding CRPS”. [12].
In the present work, the aim was to develop an effective method to separate, through Capillary
Electrophoresis (CE), and analyze digested IgG, through mass spectrometry (MS), using Electrospray
Ionization (ESI) and Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF). In
addition, the goal was to develop a protocol for the preparation of a coated capillary.
1.4. Outline
The main body of work is divided into five chapters.
It begins with the current chapter – 1. Introduction – in which the purpose of the research is presented,
as well as the goal of the study and the procedure used to achieve it.
The second chapter – 2. Background – describes the fundamentals of the three techniques used:
Capillary Electrophoresis, Electrospray Ionization Mass Spectrometry, and Matrix Assisted Laser
Desorption/Ionization Time of Flight Mass Spectrometry; along with a brief description of the protein
under study, Immunoglobulin G, and the methodology used when analyzing proteins.
The third chapter – 3. Materials and Methods – concerns the chemicals and instruments needed
throughout the experimental work. Moreover, all the preparation steps required to initialize the
experimental work and all the development of the different technical setups used are approached.
The fourth chapter – 4. Results and Discussion – presents the results obtained from the experiments
and their analysis.
The fifth and last chapter – 5. Conclusions and Future Works – is an evaluation of the obtained results.
Furthermore, it proposes several paths that should be followed as future work, aiming successful
capillary electrophoresis-mass spectrometry hyphenation.
4
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2. Background
To accomplish a successful analysis of a protein as a potential biomarker, the protein needs to be
separated and fractioned into peptides, particularly if present in complex matrices as the present case,
blood plasma. Characterization is also required to determine the molecular weight and verify the identity
of each peptide. This chapter starts with a description of the instrumentation required to apply CE and
MS as well as the theoretical concepts and relevant variables to consider during the procedure.
Additionally, an overview of the protein IgG and of the proteomics, the global scale analysis of proteins,
is presented.
2.1. Fundamentals of Capillary Electrophoresis
CE is an analytical technique used to separate components in a mixture, based on the migrations of
ionic compounds in the presence of an electric field. One of the most significant advantages of this
technique, when compared with other analytical separation techniques, such as high-performance liquid
chromatography or gas chromatography, is its applicability range [13]. It evolved from being originally
only used for analyzing biological macromolecules to being used nowadays to separate compounds of
different natures, such as amino acids, vitamins, peptides and proteins, and so on. [14]
Its wide versatility derives also from the different modes of operations of CE [14]. Capillary Zone
Electrophoresis (CZE), the mode chosen for the present work, is the simplest one. In CZE, the capillary,
which typically consists of fused silica, is filled with a background electrolyte solution (BGE), used as a
buffer, and the sample is injected at one end of the capillary.
Little instrumentation is needed as shown in Figure 1. The main components are a capillary, whose
extremities are placed in BGE vials, a high voltage supply, electrodes to connect the high voltage supply,
a detector and a computer, with appropriate software. [15]
Figure 1 - Schematic representation of CE system
Source: Sergey N. Krylov Lab http://www.yorku.ca/skrylov/research.html (adapted)
6
The sample is loaded onto the capillary by placing the inlet end of the capillary in the sample vial and
applying either an electric field (electrokinetic injection) or external pressure/vacuum (hydrodynamic
injection). The detector used is commonly an Ultra-Violet (UV) detector, placed along the capillary, which
aims to monitor how long time each species takes to move from the inlet to the outlet of the capillary.
The computer controls the running parameters and gathers the information provided by the detector, the
signal is processed and an electropherogram is generated. The electropherogram presents in the x-axis
the retention time, i.e., the time it takes an analyte to move along the effective length of the capillary,
that is, from the beginning of the capillary until it reaches the detection window. In the y-axis, the
absorbance is displayed.
The phenomenon behind CE is electrophoresis. Its definition consists of the differential movement of
charged species by attraction or repulsion in an electric field. Separation is mainly influenced by the
differences in analyte velocity when responding to the applied electric field. The charge of the ions
defines the direction of its movement: cations move toward the negatively charged cathode, anions
move toward the positively charged anode, and neutral species remain stationary, having an
electrophoretic velocity of zero. The analyte velocity, 𝑣, can be computed by eq. (2.1). [14]
𝑣 = 𝜇𝑒 𝐸 (2.1)
Where the magnitude of the applied electric field, E, is obtained by dividing the applied voltage by the
capillary length (in volts/cm). The electrophoretic mobility, 𝜇𝑒, for a given ion and medium, is a constant
of that ion. It is determined by the electric force that the molecule experiences, balanced by its frictional
drag through the medium and it is defined by eq. (2.2).
𝜇𝑒 =𝑞
6 𝜋 𝜂 𝑟 (2.2)
Where q and 𝑟 stand for the analyte’s charge and radius, respectively and 𝜂 is the BGE viscosity.
Analyzing eq. (2.1) and (2.2), one can conclude that electrophoretic mobility and, therefore,
electrophoretic velocity, decreases with the growth of the particle’s size and increases with the charge
of the analyte. [14]
Besides electrophoretic mobility, there is another phenomenon contributing to the mobility of the analyte,
the electroosmotic flow (EOF). As mentioned earlier, the capillary is typically made of silica with silanol
groups (SiOH) exposed on the inner surface. Although the “pKa of the surface silanol groups is difficult
to determine and mostly not known, in general, EOF becomes significant above pH 4.” (Lauer, Rozing,
2014) [14]. Therefore, the silanol groups deprotonate above that pH, becoming negatively charged
(SiOH→SiO-).
Cations will migrate towards the negatively charged wall to forming a narrower first layer of positive
charges (Stern layer) that neutralizes most of the silanol ions. Then, a second diffuse layer neutralizes
the remaining negative charge creating a potential difference close to the wall, the zeta potential, 𝜁. EOF
describes the movement of the electrolyte’s ions, in the diffused layer, when an electrical field is applied.
7
The magnitude of the EOF can be expressed either in terms of velocity or mobility by eqs. (2.3) and
(2.4), respectively; where 𝜀 is the solution dielectric constant.
𝑣𝐸𝑂𝐹 =𝜀 𝜁 𝐸
𝜂
(2.3)
𝜇𝐸𝑂𝐹 =𝜀 𝜁
𝜂
(2.4)
Summing the contributions of the electrophoretic mobility with the electroosmotic flow, it is possible to
define the apparent mobility. [10,12]
2.1.1. Capillary Wall Modifications
Capillary wall modifications are a method used to minimize analyte adsorption to the inner surface of
the capillary. These may cause fluctuations in the EOF and, consequently, in the migration times in the
CE experiments. Adsorption may also lead to significant band broadening, compromising the separation
efficiency.
The ultimate goal of the coating is to enhance the performance of the separation. Therefore, some
requirements need to be fulfilled. The coating should demonstrate a high “shield” capacity by cutting the
analyte-capillary wall interactions, should not interfere with the analytes and ideally be stable over time,
allowing several runs. Likewise, it should be versatile, i.e., be stable with different buffers’ nature,
concentration and pH. If possible, it should be inexpensive and both preparation and regeneration
should be simple and fast. [17]
During the last two decades, many coating techniques have been proposed. The coating process can
be done either by permanent modification with covalently bonded or physically adhered phases or by
dynamic deactivation using running buffer additives. Both approaches have been somewhat successful,
although no single method is clearly superior. However, there is not a single solution suitable for all
experiments and the procedure should be optimized for each situation.
When CE-ESI-MS is used, a dynamic coating should be excluded since it is likely to compromise the
MS detection. If the chemicals used in the coating entered the mass spectrometer, severe background
noise could occur. Other drawbacks are the suppression of analyte signals and contamination of the ion
source and MS optics [17]. Therefore, a permanent coating was applied in this project, more specifically
with polyvinyl alcohol (PVA).
The protein IgG is the object of study and, when working with proteins as analytes, analyte-capillary wall
interactions are expected to occur. PVA coating is a neutral coating and highly hydrophilic. It reduces
significantly the adsorption by suppressing the EOF, making it appropriate for protein separation [18].
This coating has also previously been determined to be highly stable over a wide range of conditions,
however its performance is enhanced under an acidic setting [19]. Moreover, it tolerates the most
common organic solvents [17].
8
2.2. Fundamentals of Mass Spectrometry
MS is an analytical technique used for a qualitative and quantitative determination. The molecules under
study are desorbed from condensed phases, and ionized when introduced into the ion source, to acquire
positive or negative charges.
In the next step, the ions go through the mass analyzer, accelerated by a strong electric field. The mass
analyzer separates the ionized analytes in line with their mass-to-charge (𝑚/𝑧) ratios. There are several
types of mass analyzers, depending on the separation method. Separation can be achieved based on
the 𝑚/𝑧 resonance frequency, 𝑚/𝑧 trajectory stability and time ions of different masses take to fly from
the ion source to the detector. Examples of each mass analyzer are Orbitrap, quadrupoles and TOF,
respectively. [20]
When the objects of study are proteins, it is common to perform tandem mass spectrometry or MS/MS.
A given ion, selected in the first mass analyzer, the precursor ion, collides and fragments, generating
so-called product ions. New 𝑚/𝑧 are then considered in a second mass analyzer. This can be performed
several times. [21] Figure 2 illustrates the several steps on the MS process.
The signals generated are showed in a computer, which displays the signals graphically as a mass
spectrum presenting their relative abundance according to their 𝑚/𝑧. The y-axis represents the intensity,
which reflects the number of ionized analytes detected and, in the x-axis, the 𝑚/𝑧. If ions are singly
charged, the value of 𝑚/𝑧 represented corresponds to its real molecular mass plus one [M+H]+.
However, if doubly charged, the real mass will be double plus two [M+2H]2+ as the output value, et
cetera. [21]
This technique presents a high versatility and adaptability due to the possibility of choosing between
several types of each component of the instrument. There are a number of different ion sources,
analyzers and detectors.
There are two types of ionization methods, the “hard” ionization, in which the analytes get highly
fragmented and the “soft” ionization in which a low degree of fragmentation occurs. Macromolecules are
usually analyzed by the last, the soft methods. Note that, in this project, the two most common soft
ionization techniques were used: Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix
Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). With the
discovery of these two soft ionization methods, the molecular weight restriction of the analytes was
Figure 2 – Schematic representation of tandem mass spectrometry
9
mitigated. Only with MALDI and ESI, it became possible to analyze large molecules (beyond 1 kDa) with
a very high ionization efficiency, revolutionizing mass spectrometry. [22]
Figure 3 presents a schematic overview of the steps occurring inside each of the mentioned instruments.
2.2.1. Electrospray Ionization
Electrospray ionization is a robust and sensitive technique that provides reliable results. The sample is
injected directly into the system. Usually, diluted in a volatile solvent solution, referred to as sheath liquid,
as an extra liquid flow that increases the total volume under analysis when low quantity of sample is
available. In this way, it is possible to conduct a more stable experiment. The sheath liquid is typically a
mixed organic/ aqueous solvent. The injection is performed with a mechanical syringe pump through a
metal capillary needle, at a low flow rate (typically 1–20 μL/min). [23]
High voltage is then applied to an electrode surrounding the capillary tip, creating a strong electrical
field. Consequently, the solution is nebulized into a charged mist. A drying gas, nitrogen, in this case, is
used not only to improve the nebulization, assisting in the droplet formation but also to direct the spray
from the capillary to the mass spectrometer. Also, this gas evaporates the solvent, decreasing the droplet
size. As the solvent evaporates, the electric field density on the droplets increases, causing similar
charges to repel each other. The point where the droplet can no longer support the surface charge is
known as Rayleigh limit. Thus, the ions detach from the droplets, moving through a heat chamber (100–
300°C) at atmospheric pressure towards the mass analyzer, that is under high vacuum. Complete
desolvation of the ions is achieved. [23, 24]
Several parameters such as drying gas temperature, flow or applied voltage can be altered during the
experiment to optimize the detection.
Figure 3 - Schematic representation of ESI-MS system (a) and of MALDI-MS (b)
Source: Nolan Speicher http://www.idtdna.com (adapted)
10
The electrospray ionization, particularly in large analytes, results in protonated or deprotonated species,
depending on the mode (positive or negative), or complexed species. For example, in positive mode,
this results in forming a range of charged species for each molecule (+2, +3, +4, etc.). As this can make
the mass interpretation rather complex, deconvolution is performed. By deconvoluting the raw data,
multiply-charged species mass is recalculated into the singly-charged mass.
For the deconvolution, the 𝑚/𝑧 and the mass (M) of each analyte needs to be known. Eq. (2.5) is used
to compute the parameters from the 𝑚/𝑧 of the multiply-charged ion. Also, the charge state (Z) must be
calculated for each ion, which is typically accomplished by using the 𝑚/𝑧 values of two adjacent peaks
in eq. (2.6). [25]
(𝑚/𝑧)𝑧 =𝑀 + 𝑍
𝑍⇔ 𝑀 = (𝑚/𝑧)𝑧 ∙ 𝑍 − 𝑍
(2.5)
𝑍 =(𝑚/𝑧)𝑧−1 − 1
(𝑚/𝑧)𝑧−1 − (𝑚/𝑧)𝑧
(2.6)
ESI is easily coupled with other instruments. Having a previous step for separation, as, for example, CE,
simplifies the analysis of the mass spectra, which can ease the interpretation of the results.
2.2.2. Matrix Assisted Laser Desorption/Ionization
MALDI-TOF MS is one of the most used mass spectrometric techniques in biological molecules studies.
This technique is an effective tool for identification of relatively pure protein samples. However, the same
cannot be said when analyzing samples with higher complexity.
In contrast to ESI, when using MALDI, the sample is not inserted directly. It is co-crystalized with an UV-
absorbing crystalline matrix material and spotted onto a metal target plate. There are several types and
fabricants of MALDI plates depending on the final application.
In this project two were used: an AnchorChipTM from Bruker Daltonics (Bremen, Germany) and a
stainless-steel plate treated in-house with Alkyl Ketene Dimer (AKD). The plates are printed with small
spots, where both sample and matrix are deposited. [21, 22]
The plate is then inserted into the instrument, where a UV laser, most commonly a nitrogen UV laser,
irradiates each spot. The matrix absorbs the energy, heating and volatilizing the sample and ionizing it
at the same time [28]. In the scientific community, the exact mechanism of ion formation does not have
a single answer. Yet, the matrix is thought to be involved in the process by providing proton donating/
accepting or electron donating/accepting species upon bombardment with the laser. [28]
The choice of a matrix has a substantial influence on the analytical value of MALDI spectra. The most
used matrices for MALDI are 2,5-dihydroxybenzoic acid (DHB); α-cyano-4-hydroxycinnamic acid
(HCCA); 3,5-dimethoxy-4-hydroxycinnamic acid (SA). The choice is based on the final purpose.
According to the manufacturer Bruker Daltonics, DHB is appropriate to analyze peptides, glycans, and
glycopeptides; HCCA for peptides and smaller proteins and SA for proteins in general. [26]
11
Generally, MALDI imparts a single charge to proteins (with an occasional +2 or +3, as well), which both
simplifies and complicates downstream analysis. Also, the mass calculation is trivial, as 𝑚/𝑧 equals the
molecular mass plus one for z=1.
2.3. Fundamentals of Immunoglobulin G
Immunoglobulins are the major class of serum glycoproteins. Found in the blood and produced by
plasma cells, immunoglobulins consist of a group of proteins built from two heavy and two light chains.
Several papers have been published detailing its functions, and how their glycan pattern can affect it
[30]. The three main purposes of immunoglobulins are to neutralize the pathogen, activate other defense
cells and activate the complement system. [35]
IgG is the most prevalent isotype present in the human body. Human IgG is divided into four subclasses
(IgG 1, 2, 3 and 4) depending on the constant regions of their polypeptide chains. Subclasses 1, 2 and
4 have a mass of roughly 150 kDa; subclass 3 has a mass of around 170 kDa. [36] Bovine IgG only has
two subclasses IgG1 and IgG2 [37]. Figure 4 presents the generic structure of IgG.
As referred previously, each IgG subclass has two light (~25 kDa), and two heavy (~50-60 kDa) identical
peptide chains. In the heavy chains there is a specific asparagine (Asn) site, at position 297, where
glycans, during glycosylation, bind to IgG. There are also other amino acids which allow different types
of glycans to bind. In human IgG, only N-linked glycosylation occurs. That is, a given glycan binds to a
nitrogen atom in an amino acid residue. [29]
The concentration of IgG in healthy human serum ranges from 7–16 g/l [38]. It could be hypothesized
that the value increases in CRPS patients, as occurs with OPG levels. In a study carried out by Krämer
et al., it is reported a significative increase of OPG levels in CRPS patients when compared with not
only healthy individuals but also with patients after uncomplicated fractures [35, 36]. The behavior of
IgG could possibly follow the same tendency.
Figure 4 – Human IgG structure
VH – Variable Heavy Chain CH – Constant Heavy Chain
VL - Variable Light Chain VL - Constant Light Chain
12
2.4. Fundamentals of Proteomics
The term proteomics is a combination of the word protein with genome and it was coined by Marc Wilkins
(1994). He defined as “the study of proteins, how they're modified, when and where they're expressed,
how they're involved in metabolic pathways and how they interact with one another". Proteomics is then
the global-scale study of proteins. [41]. There are two main approaches used in proteomics: the “top-
down” and “bottom-up” approaches.
The “bottom-up” approach has for years been the standard. This approach consists of protein
characterization by analysis of the peptides that result from its digestion. Typically, proteins are digested
chemically or with an enzyme. [35, 36]. Figure 5 illustrates a generic “bottom-up” methodology approach.
By opposition, the “top-down” method is characterized by intact protein analysis, rather than peptides,
preserving all information of the intact protein.
When comparing both approaches, the “bottom-up” reveals a higher sensitivity and versatility. In
addition, as peptides are more easily fractionated, ionized and fragmented, this approach becomes
more likely to be used in protein analysis. Potential downsides include limited protein sequence
coverage by identified peptides. This occurs since some peptides are more easily ionized that others,
outcompeting those in the ionization step. Therefore, some peptides might not be detected. Likewise,
loss of small peptide and of post-translational modifications can occur as well as ambiguity of the origin
for redundant peptide sequences. [35, 36, 37]
As mentioned previously, in a “bottom-up” approach, in the sample preparation step, proteins are
extracted and then cleaved into peptides. Several enzymes are available; however, trypsin has been
the enzyme of choice for large-scale proteomics. Advantages of using this enzyme include high stability
in a vast range of conditions and a very high cleavage specificity. Trypsin cleaves exclusively on the
carboxy-terminal side of arginine and lysine residues, except if either of them is followed by a proline
residue. In this circumstance, the cleavage will not occur. [42]
After protein digestion, peptide separation and analysis, a database search is completed to identify the
peptides. Two common databases are NCBI or Swiss-Prot. Several parameters, including the enzyme
used in the digestion step, missed cleavages, taxonomy or mass weight limit, can be manually set by
the user to refine and narrow down the search results. Special attention should be taken as this may
affect the final output.
Figure 5 – Schematic representation of the mass-spectrometry/proteomic process
13
The database search is performed by peptide mass fingerprinting (PMF). PMF consists in comparing
the theoretical peptide masses present in the database with the peptide mass list obtained
experimentally. The degree of match is quantified according to a given scoring method, depending on
the database. To be considered a solid match a certain score needs to be achieved, meaning that false
positives are not likely to be obtained. [35, 36, 37]
.
14
15
3. Materials and Methods
The current chapter presents the chemicals and instrumentation used throughout this project,
particularly in the separation and mass spectrometry. Moreover, the experimental development of the
capillary coating procedure and CE-MS hyphenation setup is presented.
3.1. Chemicals
Milli-Q water, used as a solvent for all the solutions, was purified in a Millipore Synergy 185
(Massachusetts, USA) to a resistivity of 18.2 MΩ cm at 25 ℃.
Both IgG from human and bovine serum were digested with trypsin enzyme from bovine pancreas, using
ammonium bi-carbonate salt (NH5CO3) as a buffer, all purchased from Sigma Aldrich (Stockholm,
Sweden).
In the capillary preparation step, two types of polyvinyl alcohol ((C2H4O)n) (89-98 and 30-70 kDa) and
methanol (MeOH) were used, both purchased from Sigma Aldrich. Two buffers were used in CE: a
phosphoric buffer was prepared with sodium dihydrogen phosphate (NaH2PO4∙H2O) and the pH was
adjusted with o-phosphoric acid (H3PO4); an ammonium buffer was prepared with ammonium acetate
(C2H7NO2) and the pH was adjusted with acetic acid (CH3COOH), all purchased from Merck except the
last that was purchased from Honeywell Riedel-de Haën (New Jersey, USA). Sodium hydroxide (NaOH)
was used in the capillary equilibration and obtained from Merck.
The sheath liquid in ESI was composed by, among other chemicals already referred to in this chapter,
isopropanol (C3H8O) and diethylamine (C4H11N) obtained from Sigma Aldrich.
Specific for the sample preparation step for MALDI was used acetonitrile (C2H3N), trifluoroacetic acid
(C2HF3O2), purchased from Merck. The matrices DHB and HCCA along with the peptide reference were
purchased from Bruker Daltonics.
3.2. Instrumentation
Throughout the experiment, the following instruments were used: Thermomixer and Centrifugal Vacuum
Concentrator from Eppendorf (Hamburg, Germany), Sonorex Ultrasonic Bath purchased from Bandelin
(Berlin, Germany) and a pH/Ion meter from Metrohm (Herisau, Switzerland).
16
3.2.1. Capillary Electrophoresis
Fused silica capillaries, purchased from Polymicro Technologies (Arizona, USA) were used. The outer
diameter was 375 μm and the inner diameter 50 μm. Several lengths were used with the effective length,
i.e. the length from the inlet until the UV detection window, ranging between 21 and 81.5 cm, depending
on the apparatus. All capillaries were coated permanently with a PVA solution, according to the method
used by Martin Gilges et al. [45], with adaptations described in 3.4.1. Capillary Wall Coating. The oven
used was an HP Agilent G1530A 6890 Plus Gas Chromatograph System (California, USA). The capillary
was then inserted in the Agilent CE capillary cassette.
For the IgG separation, Agilent 7100-series CE system was used, operating in the CZE mode. The
instrument was equipped with a power supply able to reach 30 kV and a real-time UV-Visible diode-
array detector (190–600 nm). Each experiment was conducted with four different wavelengths: 200,
204, 210 and 214 nm, simultaneously. Using this instrument, an electrokinetic injection was applied.
Additionally, a home-built instrument was used. This instrument was essentially composed by four
blocks: a voltage supply, the main CE box, a UV detector and the computer to collect the data. Figure 6
presents the home-built CE instrument, with the four blocks mentioned, from left to right, respectively.
The UV detector model was a 100 UV Detector from Spectra-Physics (Darmstadt, Germany). The CE
box was also connected with an air pressure line, used for hydrodynamic injection of the analyte. Inside
the CE box, there was a “vial holder” and an electrode.
Keeping the humidity of a CE environment low is crucial to suppress current dispersion. As the CE box
was not properly sealed, a flask containing a satisfactory amount of calcium chloride was placed inside
the CE box.
Signals obtained from both CE instruments were recorded with Agilent Chemstation, Hewlett-Packard
(California, USA).
Figure 6 – Setup of the home-built CE instrument (voltage supply, main CE box, UV detector and computer to collect the data, from left to right).
17
3.2.2. Electrospray Ionization
The electrospray mass spectrometry was carried out in the Bruker amaZon Speed, in positive ionization
mode. In all analysis UltraScan mode (Gauss filter of 0.080 𝑚/𝑧) was used. The software TrapControl was
used to acquire the mass spectra and DataAnalysis to process it. In this software, the signal-to-noise
ratio (S/N) of 10 was defined as the cutoff to identify a peak, that is to distinguish a peak from the
background noise. Finally, BioTools with Mascot search was used for the peptide identification. As an
input to the search, it was set that trypsin was used for digestion with up to three possible miscleavage.
A miscleavage occurs when trypsin does not cleave on a site of IgG that was prone to, during protein
digestion. Also, the taxonomy was set to Homo sapiens.
To couple the CE instrument to the ESI, a CE-MS adapter kit from Hewlett-Packard was used. This
sprayer has three orifices, one for the nebulizing gas (nitrogen), one for the CE capillary and other for
the sheath liquid.
The capillary was changed to a CE-MS cassette from Hewlett-Packard.
3.2.3. Matrix Assisted Laser Desorption/Ionization
MALDI-TOF-MS analyzes were conducted on an ultrafleXtreme system from Bruker Daltonics.
Instrument settings and mass spectra acquisition are performed in FlexControl. The laser is a Nd YAG
laser. Mass spectra were acquired in reflector mode for peptide samples, and the laser was always shot
with an intensity of at least 75%. Mass spectra were processed on flexAnalysis, S/N value of 3 for the
peak identification. Peptide identification, as in 3.2.2. Electrospray Ionization, was completed in BioTools
with Mascot search. As an input to the search, it was set that trypsin was used for digestion with up to
one possible miscleavage. Also, the taxonomy was set to Homo sapiens.
Two different plates were used: an AnchorChipTM and a stainless-steel plate treated with AKD. The spot
size was 800 and 600 μm, respectively.
The first target plate contains “anchors”, hydrophilic patches surrounded by a hydrophobic surface. This
concentrates the sample by localizing the droplets in a very small area and preventing the sample from
spreading out. The concentration effect also improves the sensitivity when analyzing dilute samples.
[26].
The second is a new concentrating MALDI target plate. The AKD creates a highly hydrophobic coating
(contact angle slightly over 130°), in a stainless-steel plate. The manufacturing process includes the
application of AKD by an airbrush, and negative contact printing to create the concentration spots [27].
In the CE-MALDI-TOF-MS hyphenation, the MALDI plate was placed on a TIXY 200 XY positioning table
from Newport Spectra-Physics GmbH (Darmstadt, Germany), where the axes X and Y were regulated.
An extended MM53M5EX Motorized MicroMiniTM Stage purchase from National Aperture (Cambridge,
UK) was used for Z-axis. An Arduino DUE (Budapest, Hungary) microcontroller along with a home-built
circuit board was used to interface the axes with a computer, controlling the deposition parameters.
18
3.3. Sample Preparation
IgG digestion was performed using the enzyme trypsin. The enzyme/ protein molar ratio used was either
1:25 or 1:37,5. Ammonium bi-carbonate salt, with a concentration of 0,1 M, was added as a buffer, to
maintain a pH of 8. The solution was left to incubate overnight, for seventeen hours at 37°C. Digestion
quenching was achieved by increasing the sample temperature to 75º C and hold it for 5 minutes. Table
1 presents the different samples used throughout the project. The list includes the IgG nature (bovine
or human), sample’s concentration, if digested or intact and the enzyme/protein ratio.
3.4. Capillary Electrophoresis
Every morning, the capillary was rinsed with Milli-Q water for 20 minutes and then conditioned with the
BGE solution for 30 minutes. A daily fresh BGE was taken from a stock solution and stock solutions
were prepared as needed or monthly. In between experiments using the same buffer, buffer
concentration and pH, the capillary was flushed with the BGE for 20 minutes. In between experiments
with different buffer solutions the capillary was flushed for additionally 10 minutes of the new BGE, i.e.
after altering the buffer the capillary was rinsed for a total of 30 minutes with BGE.
Also, before shutting down the instrument, the capillary was rinsed with the BGE solution for 20 minutes
and with Milli-Q water for 30 minutes, to remove all particles that were possibly inside it. Then, the
capillary was stored with both inlet and outlet inside a Milli-Q vial.
Unless stated differently, all injections were performed electrokinetically, with an injection time of
20 seconds and an applied voltage of 20 kV. CE runs were performed with an applied voltage of 25 kV.
Table 1 – IgG sample list
Number Nature Concentration
(mg/mL) Digested Trypsin:IgG
① Bovine 1,5 1:37,5
② Bovine 1,5 -
③ Bovine 1 1:25
④ Human 1 1:25
19
3.4.1. Capillary Wall Coating
The capillary was prepared based on the procedure described in the reference [45] with the permanent
coating. Several attempts were made in order to achieve a consistent coating method.
One of the characteristics of PVA is its high viscosity, that along with the capillary having such a small
inner diameter, makes the coating process rather challenging. To simplify the injection of the PVA into
the capillary, instead of a regular syringe that was constantly getting clogged, the metallic tool presented
in Figure 7 was used.
This tool has a hole inside where it is possible to insert the 0.5 mL Eppendorf vial with PVA, and two
orifices on top. In one of them, the capillary was inserted ensuring that the inlet was inside the vial and
the other was connected to a nitrogen line. The pressure was then set to 4 bar, forcing the PVA to pass
through the capillary.
Caution must be taken to the outlet, verifying the PVA flow. Whenever there was no flow, one of three
things occurred:
(1) The capillary was clogged. One option to solve the clogging was to rinse with water, if this did
not solve the issue then the extremities of the capillary would be trimmed;
(2) The capillary was not correctly inserted in the vial. It was necessary to open the tool and verify
the position;
(3) Leaking in the system. As this tool was only adapted to serve this purpose, there were several
spots where leaking was occurring. Rubber pieces and plastic film were added to prevent
leakages. Likewise, all nuts should be checked and leaked tight.
Complete dissolution of PVA was rather difficult to achieve, the solution had to be in the ultrasonic bath
for several hours. Two batches were made: one dissolved in cold water and another in boiling water. No
difference was observed. Two capillaries (capillaries ① and ②) were then coated, and both got
clogged, leading to the conclusion that all the small particles of PVA were not dissolved. To solve this
Figure 7 - Tool used for capillary wall coating
(a) front view; (b) top view
(a)
(b)
20
matter, the concentration was reduced to 1% in capillary ③. Clogging did not occur, however, such low
concentration showed not to be sufficient to obtain consistent electropherograms.
The next step was to change the type of PVA to a water-soluble one. With this PVA, the average
molecular mass of the polymer was the same as the one reported in [45]. In a first attempt, capillary ④
with a concentration of 5% was made. However, from the second experiment, no peaks occurred in the
electropherogram. Again, the proteins were most certainly getting attached to the capillary inner wall.
Then, in capillary ⑤ the concentration was increased to 10%, similar to the one reported in [45]. When
this capillary was tested, a step was observed in the electropherogram due to a current drop. No
regeneration of the capillary was successful.
A second capillary was prepared following the same procedure. In the first experiment with capillary ⑥,
some peaks were visible. Later trials showed no peaks. The capillary was then regenerated by rinsing
with water, methanol, and BGE for 30 minutes each. Peaks were visible once more, although the
baseline was gradually increasing along the experiment. To overcome this issue, a solution of 0,1 M of
NaOH was flushed through the capillary for 5 minutes. This procedure was followed throughout the
project, every time needed. Note that, as the capillary is coated with PVA, only low concentrations should
be used not to damage the coating. Figure 8 illustrates the impact of rinsing the capillary with NaOH.
Afterwards, consistent results and stable current were obtained. For this reason, capillary ⑥ was used
in the initial CE experiments. After those, ESI-MS was performed. During that period, both capillary ends
were kept in water. Before coupling both instruments, the capillary was tested and identical
electropherograms were obtained. Therefore, the capillary was adapted to CE-ESI-MS, as described in
the chapter 3.5.1. CE-ESI-MS Hyphenation. After some experiments the capillary was damaged, so a
new capillary had to be prepared to be used in the CE-MALDI-MS apparatus.
Capillaries ⑦ to ⑭ were prepared using the same method as for ⑥. With capillary ⑦, no consistent
results were obtained. Capillaries ⑧ to ⑫ broke in half during the experiments with no clear cause
detected. Capillary ⑬ clogged overnight. Finally, capillary ⑭ showed characteristics suitable to be used
in the CE-MALDI-MS experiments. Relevant capillaries’ parameters can be found in Table 2.
Figure 8 – CE electropherogram illustrating the impact of rinsing the capillary with NaOH (a) before rinsing the capillary with NaOH; (b) after rinsing the capillary with NaOH
21
Table 2 - Capillary parameters, wall coating procedure, symptoms and setup used
Number PVA MW*
(kDa) PVA conc.
(% w/w) Solvent
Total/eff. length (cm)
Symptom Setup
① 89-98 5 Cold water 80/71,5 Capillary clogged. CE
② 89-98 5 Hot water 75/66,5 Capillary clogged. CE
③ 89-98 1 Cold water 75/66,5 Current dropped during the first run. After trimming
capillary ends, current back but results not consistent. CE
④ 30-70 5 Cold water 90/81,5 1st run working well, then no more peaks. CE
⑤ 30-70 10 Cold water 90/81,5 Current dropped during the first run. CE
⑥ 30-70 10 Cold water 88/79,5
88/40
1st run working well, then no more peaks. After rinsing with
the BGE, there were peaks again but with an inclined
baseline. Rinsed with NaOH (0,1 M). Afterwards,
consistent results and stable current. Very low EOF.
Damaged in ESI.
Used from 26.03.2018 to 25.04.201 8.
CE
CE-ESI
⑦ 30-70 10 Cold water 80/21 No consistent electropherograms. CE-MALDI
⑧ - ⑫ 30-70 10 Cold water 80/22 Broken during CE experiment. CE-MALDI
⑬ 30-70 10 Cold water 77,5/21 Capillary clogged overnight. CE
⑭ 30-70 10 Cold water 87,5/24 Consistent results and stable current. Very low EOF.
Used from 25.06.2018 CE-MALDI
*MW represents mass weight.
22
Whenever a new capillary was prepared, after 4 hours in the oven at 140º C, the capillary was flushed
using the following steps:
(1) MeOH for 20 min;
(2) Milli-Q water for 20 min;
(3) BGE for 30 min.
The effectiveness of the coating was tested using mesityl oxide. This component is neutral and therefore,
theoretically, no evident peaks should occur. As there was only one peak after almost one hour, one can
conclude that the PVA wall coating was effective, as the EOF was very low. The electropherogram
obtained is presented in Figure 9.
Throughout the development of a PVA coating capillary method, it was noted that wetting the capillary
with water before injecting the PVA, decreased the probability of clogging.
3.5. Electrospray Ionization
Three different types of sheath liquid were used: 50/50 (v/v) water/methanol with 5 mM ammonium
acetate; 50/50 (v/v) water/isopropanol with 5 mM ammonium acetate and 50/50 (v/v) water/isopropanol
with 5 mM ammonium acetate and 1,5% (v/v) of diethylamine. This last component was added to
decrease the ions charge state [46].
Before all analysis, the helium line, used in the ion trap, was flushed 2 times to guarantee the presence
of helium. Also, the syringe used in the mechanical syringe pump was cleaned by flushing several times
with acetonitrile, Milli-Q water, and isopropanol. Then, the same chemicals were flushed through the
system.
A blank experiment was performed to verify the background noise and potential contaminations. In case
of detection of contaminants, thoroughly cleaning would be executed by extensive flushing.
Blank and sample solutions were always ultra-sonicated for 10 minutes before the injection into the
instrument. IgG was analyzed with a concentration of 15 μg/mL in a solution with a volume of 20 μL.
Figure 9 – CE electropherogram of mesityl oxide 8,5 mg/mL; injection: 20 kV for 20 s; voltage: 25 kV;
buffer: sodium phosphate (pH=3.2; 100 mM)
23
3.5.1. CE-ESI-MS Hyphenation
In order to be used in the CE-ESI-MS hyphenation, the capillary had to be adapted. First, a new window
had to be made leaving the capillary with one window at 8.5 cm (CE) and a second at 45 cm (CE-ESI).
Extra caution had to be taken when handling the capillary and inserting it inside the cassette, as the
windows are especially fragile.
Second, the outlet tip, that was inserted in the CE-MS sprayer adaptor kit, needed to be flattened. The
precision of the spray depends on the quality of this cut. The cut itself, as all previous capillary cuts, was
made with a CE column cutter, and then flattened with a thin sandpaper. The apparatus shown in Figure
10 was used to verify the flatness of the capillary tip.
Finally, the capillary should protrude approximately 0.1 mm out of the sprayer tip. This length was
adjusted with the screw of the sprayer tip and then optimized by checking the actual length in the
computer screen. Then the cassette was carefully placed in the instrument to avoid any potential
deviations in the position.
Figure 11 presents the set-up used for this part of the study. On top it is possible to observe the entire
set, with the computer that controls the CE on the left and the ESI computer on the right. The height of
the capillary inlet was adjusted to be leveled with the MS spray tip outlet to avoid siphoning effects. The
instruments should also be kept as close as possible to minimize the total capillary length needed and
shorten the sample “travel” time along the capillary.
Figure 10 – Apparatus used to verify the cut of the capillary (a) detail on the capillary tip; (b) detail on lenses used; (c) overview of the setup
(a) (b)
(c)
24
3.6. Matrix Assisted Laser Desorption/Ionization
External calibration was performed before every analysis, using peptide calibration samples (Peptide
calibration standard) supplied by the manufacturer Bruker Daltonics. Additionally, internal calibration
was performed using the 𝑚/𝑧 of the autodigestion of the trypsin.
Samples were acidified with trifluoroacetic acid and applied to the MALDI target plate. DHB and HCCA
matrices were prepared according to the recommendations of the manufacturer [26].
The TA30 washing solution was prepared by mixing 30% acetonitrile together with 70% of
0.1% trifluoroacetic acid solution. A fresh solution was prepared for each experimental run. A solution of
DHB powder with a concentration of 20 mg/mL in TA30 was used. The DHB matrix solution (20 mg/mL
2,5-DHB in TA30) was vortexed until it became transparent, indicative of complete dissolution.
A volume of 0.5 µL of the sample solution (1 mg/mL IgG) was deposited onto each MALDI target plate
position. Then, after total solvent evaporation, 0.5 µL of the matrix solution was deposited on top of the
sample. The same procedure was applied to the peptide calibrants. Four replicate spots were used for
each sample and calibrant. The plate was then ready to be inserted in the instrument.
3.6.1. CE-MALDI-TOF-MS Hyphenation
The CE instrument was coupled off-line with the MALDI-TOF-MS instrument by using a positioning table
and a robotized arm (X-Y axes, and Z axis, respectively). The software allowed selecting several lineups
such as, deposition along a single row, deposition along a single column, or even customizable patterns.
The height of the robotic arm when changing from one spot to the next as well as the time of deposition
Figure 11 – Setup used for CE-ESI-MS analysis
25
on each anchor could also be chosen. The control program in Arduino, available from previous works
[27, 47], was adapted to the present work.
Several modifications were completed to improve the experimental setup. Initially, the capillary outlet
along with an external electrode were introduced into a vial, filled with the BGE solution, located on the
positioning table.
By analyzing the sample in CE, before hyphenation, the time that a given analyte takes to move from
the inlet until the detection window is known. Then, the minute in which the analyte should reach the
capillary outlet was computed. This calculation was done taking into account the effective and the total
length of the capillary, assuming that the electrophoretic mobility is constant. The following step was to
insert this information as an input to the Arduino platform that controls the deposition on all axes.
Changing spots would occur as follows: the robotic arm would raise the capillary, move to the following
spot and then lower the position. The capillary height was set to increase 2 mm, to ensure that the
droplet would not be dragged in the process.
The CE software was programmed, in a function named “Timetable” to change the applied voltage to
zero, 30 seconds before the first relevant analyte reached the capillary outlet and then back to the initial
voltage. This voltage interruption was meant to allow the user to manually move the separation capillary
from the vial to the starting spot on the MALDI plate, inserting it and fixing onto the robotic arm. Then,
the Arduino would be set to start the deposition according to the selected program.
As there was no significant EOF, a second capillary was used to create a “sheath liquid effect”,
generating a spray in the separation capillary tip and dragging the sample to the MALDI target plate.
In a first attempt, the second capillary had one of the extremities in a vial filled with BGE and the other
connected to the separation capillary, with a small plastic tube. A pressure of 1.5 bar was applied to
force the BGE through the second capillary. An electrode was also positioned in the vial. During the
separation, the pressure was always being applied. Note that this vial was grounded to the MALDI plate.
26
Figure 12 illustrates the experimental setup ①. The vial with the second capillary can be distinguished
by the red lid.
A second mode of operation was created to address two obstacles of the experimental setup ①. The
first was the fact that part of the liquid was rising along the outside of the capillaries and not being
deposited onto the plate. The second, setup ②, was related to the capillary being too short to reach the
MALDI plate, after breaking during one run. A solution was found in using a plastic piece with four orifices
and with a minor dead volume in the middle. A different capillary was inserted in each one of three
orifices and the remaining was covered.
Other modified was to remove the electrode inserted in the vial used to create the spray. For several
experiments the current was decreasing abruptly after a few minutes, the cause was not understood.
However, when the electrode was removed from the setup, a stable current was achieved.
With this mode of operation, the capillary was not initially in a vial but placed onto a spot in the MALDI
plate. Before starting the separation, a droplet of BGE was deposited in the spot to ensure electrical
contact during the process.
In contrast to the previous scenario in which the pressure was constantly applied, in mode ② the
pressure had to be disabled whenever the droplet size was too large to fit in one spot, to prevent
contamination in other spots.
Figure 12 – CE-MALDI-MS setup ①
(a) capillary used for the separation; (b) capillary used to create the spray effect
(c) vial filled with BGE where the capillary used to create the spray effect is inserted
(c)
27
In Figure 13, the experimental setup ② is presented.
Figure 13 – CE-MALDI-MS setup ②
(a) capillary used for the separation; (b) capillary used to create the spray effect
28
29
4. Results and Discussion
4.1. Capillary Electrophoresis
In this chapter the most relevant results from the experiments that were done are presented as well as
its analysis. It is divided in four sections, starting with the results from the bovine IgG separation using
CE. The second section is concerning human IgG analysis through ESI stand alone and coupled with
CE. Likewise, MALDI results are presented as well as the results from the off-line CE hyphenation
attempts. This chapter ends with the fourth section in which a brief comparison between both mass
spectrometry techniques is presented. Capillary electrophoresis was used to separate the protein IgG
into peptides. Initially, bovine IgG samples were used, given its similarity with human IgG and its lower
cost when compared with human IgG. Before performing the separation, the sample was digested with
trypsin left to incubate overnight.
The first CE investigation was done to understand the behavior of the sample when injected. The first
sample prepared, sample ①, had a concentration of 1.5 g/mL of trypsin digested bovine IgG, prepared
as described in chapter 3.3. Sample Preparation. The electropherograms presented in Figure 14 were
all obtained using sample ①, and conditions, leaving the vial inside the instrument during the four runs
as well as the rinsing step with the BGE between runs. Note that the initial conditions were chosen
based on non-published studies done with the same instrument and type of sample. Electropherograms
were labeled chronologically.
All electropherograms henceforth presented are collected registering a wavelength of 214 nm unless
stated.
Observing the electropherograms it is possible to see that the results are not reproducible, even though
no parameters were altered in between runs. In the first electropherogram, A, around 40 minutes, there
is a large hump suggesting incomplete digestion of the IgG. In more detail, the electropherogram of an
intact protein is characterized by one single broad peak. As for the case of a digested protein, multiple
peaks are expected, each corresponding to a given peptide. To test this hypothesis, intact bovine IgG
(sample ②) was analyzed, resulting in the electropherogram presented in Figure 15. The fact that the
peak of the intact protein occurs around the same time supports the hypothesis of incomplete digestion.
30
The last three electropherograms illustrated in Figure 14, B, C and D, show a different behavior, not
showing the large hump. In opposition, around 40 minutes, a cluster of peaks is observed. The high
number of peaks may indicate that the sample is somehow changing. As the sample is being analyzed
under the same conditions, it was expected to obtain replicates of the first electropherogram. However,
the large hump in A is evolving to several peaks in B, C and D. Possibly the quenching of the digestion
was not successful, though no conclusions on the cause of this cluster of peaks can be drawn.
Nevertheless, excluding the systematic peak at ~1 min and the group of peaks after 40 min, and
zooming in in the remaining, it is possible to observe almost all the same peaks in the four
electropherograms of Figure 14.
Figure 15 – CE electropherogram of intact bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s;
voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)
Figure 14 - CE electropherogram of digested bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s;
voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)
31
That is shown in Figure 16, where electropherograms B and D (from Figure 14) are presented as an
example of these similarities. The peaks were labeled to facilitate the comparison between
electropherograms, according to their shape and retention time. The labels are arbitrary and do not
correspond to any identified substances. Groups A and B correspond to peaks that could not be
numbered given their inconsistency in between electropherograms.
The next step was an optimization of the buffer concentration. Figure 17 presents the result obtained
when the concentration of the buffer decreases from 100 mM to 50 mM. On one hand the peak intensity
decreases (Figure 17, top panel), but on the other the resolution of the electropherogram improves as
the peaks are better separated (Figure 17, bottom panel).
Figure 17 - CE electropherogram of digested bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s;
voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 50 mM)
Figure 16 - CE electropherogram of digested bovine IgG 1,5 mg/mL; injection: 20 kV for 20 s;
voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)
32
By observing Figure 17 it is also possible to see that the retention time was shorter than in the previous
case, as the corresponding peaks occurred in a shorter time.
To improve digestion efficiency, the concentration of IgG was decreased to 1 mg/mL (sample ③), while
maintaining the same number of moles of trypsin. As expected most of the peaks that occurred in the
previous sample were also present in sample ③, as seen in Figure 18.
After 35 minutes, the behavior becomes quite different when compared with the previous
electropherograms. Peaks from number 22 to 28 seem to be missing and many more peaks appear
after minute 40, suggesting that the tryptic digestion was more effective.
At this point, based on the results presented in Figure 18, it can be concluded that. a proper method to
separate the digested IgG was established. The method was tested by analyzing three times the sample
under the same conditions and reproducible results were achieved. Then, the goal was to couple the
separation step to mass spectrometry, beginning with ESI. However, transferring a CE-UV method to
CE-ESI-MS, is not a straightforward task and special attention must be given to the method’s
compatibility with MS requirements.
In this project, the main constraint was to find an appropriate BGE that suited both the PVA coating of
the capillary, and that was constituted of volatile components, to avoid any contamination of the MS by
non-volatile salts and high background signals. Due to the last restriction, sodium phosphate could not
be an option.
Both ammonium acetate and formate are generally used in ESI, since they evaporate readily as a
volatile component. In this case, ammonium acetate was chosen as ammonium formate could interfere
with the coating. Several pH values and different buffer concentrations were tested to reach the optimal
conditions. The first attempt was to use the same conditions as in the previous buffer and this is
presented in Figure 19.
Figure 18 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s;
voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)
33
The baseline obtained in the previous electropherograms was highly unstable. To improve that, an
attempt with a higher pH (4.6) was carried out. The result can be observed in Figure 20. However,
increasing the pH did not improve the stability of the baseline.
A flatter baseline was only achieved when the concentration was decreased to a quarter of the initial,
i.e., 25 mM. The parameters with the best results were seen at a concentration of 25 mmol/L and pH 4.2,
used in Figure 21.
Despite the optimization attempts, the electropherograms using this buffer were much less resolved
than with sodium phosphate, and presented much fewer peaks. Further optimization could be carried
out, in the future, when performing CE-ESI-MS experiments.
Figure 21 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s; voltage:
25 kV; buffer: ammonium acetate (pH=4.2; 25 mM)
Figure 20 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s; voltage:
25 kV; buffer: ammonium acetate (pH=4.6; 100 mM)
Figure 19 - CE electropherogram of digested bovine IgG 1 mg/mL; injection: 20 kV for 20 s; voltage:
25 kV; buffer: ammonium acetate (pH=3.2; 100 mM)
34
4.2. Electrospray Ionization
Before coupling the instruments, ESI was optimized separately. CE-ESI-MS can be used via a sheath–
liquid or a sheathless interface, but in proteomics studies a sheath–liquid interface is mostly used. As
previously mentioned, three different sheath liquid solutions were tested: 50/50 (v/v) water/methanol,
50/50 (v/v) water/isopropanol and 50/50 (v/v) water/isopropanol and 1.5% (v/v) of diethylamine, all
solutions with 5 mM ammonium acetate. Three mass spectra were obtained and compared in terms of
resolution, peak shape, S/N and charge state, but no relevant differences were found. Thus, 50/50 (v/v)
water/methanol with 5 mM ammonium acetate was chosen for being the simpler sheath liquid of the
three tested.
Figure 22 shows the mass spectrum of human IgG with a concentration of 15 μg/mL.
The data present in the mass spectrum was searched against a protein database, in this case, using
Mascot, but no significant matches were found. Therefore, the experimental peak list was compared
with a theoretical trypsin digested IgG mass list, present in the appendix B. List of Digested Peptides
from IgG. More specifically, the list includes the four types of IgG and a maximum charge state of +4
was assumed. Additionally, the peak list was compared with a contamination list available in the
reference [48], in which most common MS contaminants are specified.
A S/N of 10 was set as the cutoff to identify a peak. Table 3 presents the most relevant peaks
corresponding to the mass spectrum in Figure 22, that is, peaks which 𝑚/𝑧 matched either a
contamination or the theoretical IgG digestion, with a difference of 𝑚/𝑧 0.2 or lower. Additionally, the
table presents the intensity, S/N of each peak as well as mass and charge of the possible matches.
Figure 22 - ESI-MS spectrum of digested human IgG 15 𝝁g/mL; scan mode: UltraScan; flow rate:
2 L/min; capillary voltage: 4 kV; dry gas flow: 6 L/min; dry gas temperature 300 °C; nebulizer
pressure: 1 bar; mass tuning: 150 to 1500 m/z; target: 800 m/z
35
Table 3 – ESI peak list of digested human IgG 15 𝝁g/mL and comparison with theoretical data
Experimental 𝒎/𝒛
Intensity S/N Contamination IgG type Charge
state
Theoretical mass
(Da)
353.17 376674 55.0 Triton - - 353.27
375.16 239256 34.9 - 1,3,4 +4 375.21/1497.85*
447.25 294566 43.0 - 1,2,3,4 +1 447.25
448.24 352608 51.5 - 1,2,3,4 +1 448.28
449.20 239726 35.0 - 2 +4 449.25/1793.99*
679.35 285981 41.7 PEG - - 679.41
787.64 239792 35.0 - 1
3,4
+2
+4
787.43/1573.86*
787.65/3147.58*
844.82 206832 30.2 - 3,4 +4 844.65/3375.60*
Note that peaks that were not allegedly matched with the theoretical nor with the contamination peak
list, were removed from the table. Also, all the values hereafter signalized with “*” represent the mass of
the singly charged peak, computed by the eq. (2.5).
From the mass spectrum (Figure 22), it is possible to observe six peaks with a molecular mass similar
to the theoretical trypsin digested IgG mass. One peak also presented a mass identical to the polymer
polyethylenglycol (PEG) and other to triton, a substance that is usually present in detergents.
The peaks possibly matching the contaminations (𝑚/𝑧 353.17 and 𝑚/𝑧 679.35) were not further
investigated, as it is not the purpose of the study. In opposition, and given that with only the information
present in Table 4 and in the mass spectrum in Figure 22 it is not possible to conclude if the peaks are
in fact from IgG, an analysis over each peak was carried out. Figure 23 presents a zoomed in view of
the six potentially related to IgG peaks. Unfortunately, it is possible to note a very poor and broad shape.
To verify the charge state of a given ion, the isotopic pattern is analyzed. In proteins, the most
predominant isotope is 13C, that is, carbon with one extra neutron, with a molecular mass of around
1 Da. When a given ion with mass M is singly charged, a second peak, the isotopic peak, should be
present at 𝑚/𝑧 ([M+H]++1)/1.
Following the same principle, if an ion is quadruply charged, the isotope peak should be detectable by
a 𝑚/𝑧 distance of 0.25. Analyzing the first peak (𝑚/𝑧 375.16) in Figure 23 (a), it is not possible to
observe the isotopic pattern, and therefore this peak is not be related to the theoretical one.
The next three peaks on the list are seen in Figure 23 (b). With such a broad peak, the analysis is rather
complex. All three peaks have a distance of roughly 1 𝑚/𝑧, possibly a singly charged isotopic pattern.
If 𝑚/𝑧 448.24 is indeed from the isotope of 𝑚/𝑧 447.25, then the last would be the only peak, of this
two, belonging to the peptide. The same follows to the next pair (𝑚/𝑧 448.24 and 𝑚/𝑧 449.20). If the
second peak is an isotope of the first, then only 448.24 is related to the listed IgG peptide mass. Also,
with such low intensities, the hypothetical isotopic pattern could be result from the noise.
36
However, to verify the isotopes existence, the presence of the peak at a certain 𝑚/𝑧 is not enough. It is
also necessary to verify the area of the allegedly isotopic peaks. The peak area is often interchanged
with the peak intensity, however as all three peaks are overlapping such analysis is not possible. That
said, it cannot be concluded that this ion is from IgG. Nevertheless, as the peak 𝑚/𝑧 447.25 is present
in several runs performed (including in the mass spectrum of Figure 26, even though with a very low
S/N), it is a possible match.
For the peak 𝑚/𝑧 787.64, two different possible matches were found. However, by analyzing the
zoomed in mass spectrum, this value is seen at a too high 𝑚/𝑧 to correspond to the “right” peak. This
peak is then excluded from the possible match list. The same follows with the last peak. In Figure 23 (d)
there are too many peaks overlapping and again, the peak listed is seen at a too high 𝑚/𝑧.
Consequently, this peak was also excluded.
Besides too few peaks matching the theoretically digested IgG, the contaminations detected represent
a concern to the quality of the results. To eliminate the contaminations, the solvent containers as well
as the syringe were cleaned thoroughly.
A second study was performed, using the same IgG stock solution. Figure 24 presents the mass
spectrum of human IgG with a concentration of 15 μg/mL. Table 4 presents the peak list corresponding
to the mass spectrum in Figure 24 along with the possible peak identification.
373.89
375.161+
376.021+
+MS, 7.2-8.7min #263-332
0.0
0.5
1.0
1.5
2.0
5x10
Intens.
371 372 373 374 375 376 377 378 379 380 m/z
447.25
448.391+
449.201+
+MS, 7.2-8.7min #263-332
0
1
2
3
5x10
Intens.
443 444 445 446 447 448 449 450 451 452 m/z
843.711+
844.401+
845.551+
846.251+
846.881+
+MS, 7.2-8.7min #263-332
0.75
1.00
1.25
1.50
1.75
2.00
5x10
Intens.
841 842 843 844 845 846 847 848 849 850 m/z
785.451+
786.751+
787.641+
789.011+
+MS, 7.2-8.7min #263-332
1.0
1.5
2.0
5x10
Intens.
783 784 785 786 787 788 789 790 791 792 m/z
Figure 23 – Zoomed in view of the peaks potentially related to IgG
(a) 𝒎/𝒛 375.16; (b) 𝒎/𝒛 447.25, 448.24, 449.20; (c) 𝒎/𝒛 787.64; (d) 𝒎/𝒛 844.82
(a) (b)
(c) (d)
37
Regarding the contaminations, triton was successfully eliminated, as opposed to PEG, for which the
peak 𝑚/𝑧 678.34 is still present. Figure 25 presents a zoomed in view of the first three peaks listed in
Table 4.
Table 4 – ESI peak list of digested human IgG 15 𝝁g/mL and comparison with theoretical data
Experimental
𝒎/𝒛 Intensity S/N Contamination IgG type
Charge
State
Theoretical mass
(Da)
429.76 526886 20.3 - 1,2,3,4 3 429.56/1286.67*
625.73 383548 14.8 - 3,4 4 625.80/2500.17*
678.34 1205877 46.5 - 1,2,3,4 1 678.36
679.38 1203604 46.4 PEG - - 679.41
Figure 24 - ESI-MS spectrum of digested human IgG 15 𝝁g/mL; scan mode: UltraScan; flow rate:
2 L/min; capillary voltage: 4.5 kV; drying gas flow: 6 L/min; drying gas temperature 300 °C; nebulizer
pressure: 0.9 bar; mass tuning: 300 to 1000 m/z; target: 500 m/z
429.762+
430.412+
+MS, 8.9-9.6min #435-479
1
2
3
4
5
5x10
Intens.
427 428 429 430 431 432 433 434 435 m/z
625.73 +MS, 8.9-9.6min #435-479
1.0
1.5
2.0
2.5
3.0
3.5
5x10
Intens.
622 623 624 625 626 627 628 629 630 631 m/z
678.342+
679.022+
679.382+
+MS, 8.9-9.6min #435-479
0.2
0.4
0.6
0.8
1.0
1.2
6x10
Intens.
675 676 677 678 679 680 681 682 683 684 m/z
Figure 25 – Zoomed in view of the peaks potentially related to IgG
(a) 𝒎/𝒛 429.76; (b) 𝒎/𝒛 625.73; (c) 𝒎/𝒛 787.64; (d) 𝒎/𝒛 678.34
(a) (b)
(c)
38
Regarding the peak 𝑚/𝑧 429.76, represented in (a), one possible match was found. The theoretical
peak is triply charged, and therefore an isotopic pattern of a triply charged ion should be observed to
confirm this match. In fact, it can be observed in Figure 25 (a), with a smaller peak roughly at 𝑚/𝑧
430.09 and other at 430.41, separated by about 𝑚/𝑧 0.33. Concerning the peak intensity as all three
peaks (𝑚/𝑧 429.76, 430.09 and 430.41) are overlapping such analysis is not possible. Therefore, it
cannot be concluded that this ion is from IgG.
Concerning the next peak, 𝑚/𝑧 625.73 is apparently a good match with IgG. However, zooming in, it is
evident that the S/N is too low to confirm (Figure 25 (b)). Additionally, no possible isotope peaks were
found, that is, at 𝑚/𝑧 [M+H]++0.25. Also, with a high charge state it is also likely to find the same peak
at lower charge stages and that did not occur. Given that, this peak is not likely to be a match.
In the third mass spectrum, Figure 25 (c), 3 main peaks can be observed: 𝑚/𝑧 values of 678.34, 679.02
and 679.38. The one possibility of match with the singly charged theoretically digested IgG would be if
𝑚/𝑧 679.38 was an isotope (roughly 678.34+1) and the peak in between those was related with different
ion. Moreover, the possible isotope has the same intensity as the main peak leading to the conclusion
that this is not likely to be a match.
A third experiment was carried out as an attempt to improve the number of IgG peaks identified, the
peak resolution and to mitigate all contaminations. Sample concentration was increased from 15 𝝁g/mL
to 100 𝝁g/mL. Figure 26 presents the mass spectrum of digested human IgG and Table 5 presents the
most relevant peaks along with the comparison with the possible matches.
Table 5 – ESI peak list of digested human IgG 100 𝝁g/mL and comparison with theoretical data
Experimental
𝒎/𝒛 Intensity S/N IgG type Charge state
Theoretical mass
(Da)
340.20 283824 14.3 1,2,3,4 2 339.68/678.36*
447.10 91935 4.6 1,2,3,4 1 447.26
678.95 797889 40.3 1,2,3,4 1 678.36
Figure 26 – ESI-MS spectrum of digested human IgG 100 𝝁g/mL; scan mode: UltraScan; flow
rate: 2 L/min; capillary voltage: 4 kV; drying gas flow: 4 L/min; drying gas temperature 300 °C;
nebulizer pressure: 0.9 bar; mass tuning: 200 to 1500 m/z; target: 800 m/z
39
In similarity with the previous cases, the zoomed in peaks are presented in Figure 27.
In this mass spectrum, two peaks possibly from the same ion were detected: the singly charged
𝑚/𝑧 678.95 and the doubly charged 𝑚/𝑧 340.20. The mass difference of the singly charged ion is rather
high when compared to the theoretical peak (0.69 Da). Nevertheless, the existence of two peaks with
corresponding masses of different charge states is a strong indication for this match. Although it is not
possible to confirm, it is likely to be a positive match.
The peak 𝑚/𝑧 447.10 was previously analyzed, as it is also present in the mass spectrum of Figure 22.
In order to improve the mass spectra resolution, and once no contaminations were detected, the
following step was the hyphenation of this technique with the capillary electrophoresis.
4.2.1. CE-ESI-MS Hyphenation
After coupling the two instruments, several contaminations were visible in the mass spectra. The largest
concern was the PVA contamination. As a permanent coating, PVA was not expected to reach the mass
spectrometer, since it meant that the coating was being dragged from the capillary. Besides the possible
interferences in the MS, the proteins also seemed to be absorbed to the capillary inner walls.
In addition, the hyphenation itself was not successful given that when a given analyte was separated it
did not reach the mass spectrometer detector. In more detail, knowing the retention time in which a
given peak is visible in the UV-detector, the capillary length from the inlet to the UV-detector and from
447.10 +MS, 9.8-15.5min #699-1096
2
4
6
8
4x10
Intens.
443 444 445 446 447 448 449 450 451 452 m/z
678.95+MS, 9.8-15.5min #699-1096
0
2
4
6
85x10
Intens.
675 676 677 678 679 680 681 682 683 m/z
340.201+
340.63341.07
1+
+MS, 9.8-15.5min #699-1096
0.0
0.5
1.0
1.5
2.0
2.5
5x10
Intens.
336 337 338 339 340 341 342 343 344 345 m/z
Figure 27 – Zoomed in view of the peaks potentially related to IgG
(a) 𝒎/𝒛 340.20; (b) 𝒎/𝒛 447.10; (c) 𝒎/𝒛 787.64; (d) 𝒎/𝒛 678.95
(a) (b)
(c)
40
there to the outlet, and assuming that the electrophoretic mobility is constant, it is possible to determine
when it should be visible in the mass spectrometer detector.
The two reasons listed above: the capillary coating being damage, requiring to be replaced and the
hyphenation not being fully accomplished led to delays on this project. As this study was time-limited,
the decision was taken to try a different mass spectrometer, the MALDI-TOF. Therefore, as future work
further studies using the CE-ESI-MS hyphenation are suggested.
4.3. Matrix Assisted Laser Desorption/Ionization
Following the same optimization steps used in the ESI experiments, before testing the hyphenation, the
IgG samples were analyzed in MALDI to determine the 𝑚/𝑧 of each peptide, with no preceding
separation. The method was calibrated before each analysis with a peptide calibration standard.
Additionally, internal calibration was performed using the 𝑚/𝑧 ratio of peaks from the autodigestion of
the trypsin.
Figure 28 presents the MALDI-MS mass spectrum of trypsin digested IgG. A 50/50 sample/DHB matrix
mixture was deposited in each spot, on an AnchorChipTM MALDI plate.
The resultant data was then searched against a protein database to identify the peptide sequences and
further infer the protein content of the sample. As mentioned previously, there are four types of human
IgG. From these, only IgG 1 had a score over 56, the value required to positively identify a given protein.
Nevertheless, IgG 2, 3 and 4 also gave high scores, as observed in Figure 29.
Figure 28 - MALDI-TOF-MS spectrum of digested human IgG; matrix: DHB; shot at 90% laser
intensity (1000 shots per burst, 6000 shots total), pulsed ion extraction by reflectron
41
Sequence coverage was 39.4% for IgG 1, 27.3% for IgG 2, 25.7% for IgG3 and finally 26.6% for IgG 4.
More detailed information can be found at appendix C. Biotools Analysis. Table 6 presents the peak list
from the MS spectrum as well as the theoretical masses. Besides IgG, masses corresponding to bovine
trypsin and human keratin were also found.
Table 6 - MALDI peak list of digested human IgG and comparison with theoretical data
Experimental
m/z
Theoretical mass
(Da)
IgG
type Other
Experimental
m/z
Theoretical mass
(Da)
IgG
type Other
824.47 824.49 2 - 1677.84 1677.80 1 -
830.45 830.46 4 - 1690.86 1690.90 1 -
835.42 835.43 1,2,3,4 - 1794.02 1793.99 2 -
838.49 838.50 1,3 - 1808.03 1808.01 1,3,4 -
906.50 906.50 - Trypsin 1896.04 - - -
1020.51 1020.50 - Trypsin 1904.96 1904.94 2,3 -
1104.61 1104.61 1,2,4 - 1905.98 1905.89 2 -
1111.57 1 111.56 - Trypsin 1920.96 1920.94 2,3 -
1153.58 1153.57 - Trypsin 2082.03 2082.01 1 -
1172.61 1173.52 3,4 - 2163.07 2163.06 - Trypsin
1186.64 1186.65 1 - 2214.17 2214.19 2 -
1230.63 1230.63 2,3,4 - 2229.18 2228.21 1,3,4 -
1264.66 1264.66 1,3 - 2273.15 2273.16 - Trypsin
1364.66 - - 2545.05 2544.13 1,4 -
1365.68 1365.64 - Keratin 2550.14 2550.23 - Trypsin
1433.74 1433.72 - Trypsin 3773.79 - - -
Figure 29 - Results obtained from Biotools database search for MS analysis (database: Swiss-Prot)
42
After the determination of the 𝑚/𝑧 ratio of each peptide and analyzing the ones of interest, further
fragmentation was performed. The results from MS/MS mass spectrometry are presented in Figure 30.
Note that all MS/MS spectra were analyzed simultaneously in Mascot software. A score of 100 was
achieved for IgG 1. However, only 4 of the 11 peaks initially identified in the MS spectrum were positive
to IgG 1 in this database search. This could be due to the lower mass accuracy of tandem MS when
compared to MS or it could also mean that the remaining peaks were, in fact, false positives.
Figure 31 illustrates the information obtain from the Biotools database search for a given percursor ion,
in this case for 1186.65 Da.
Figure 30 - Results obtained from Biotools database search for tandem MS analysis (database: Swiss-Prot)
Figure 31 – Results obtained from Biotools database search for tandem MS analysis of peak
1186.65 Da (database: Swiss-Prot)
43
CE-MALDI-TOF-MS Hyphenation
Originally, the idea was to perform the hyphenation using the home-built CE instrument. As it is home-
made, it can be adapted so that it accomplishes the requirements. In this specific case, this instrument
had the advantage of allowing the user to apply voltage and pressure simultaneously, something that is
not possible using the commercial CE instrument. That said, the separation could occur without
interruptions and the pressure could guarantee a constant flow from the capillary inlet until the MALDI
plate.
After connecting all the parts of the home-built instrument (voltage supplier, main CE box and UV
detector) and inserting a test capillary, a very unstable current was detected. The connection with the
electrodes could be a potential issue, however after verifying it no improvements were obtained. Then,
all the parts were exchanged, one at a time, and the current was still not stable. It was concluded that,
as the CE box was not properly sealed together with the high humidity present is the air, it was not
possible to proceed used this instrument. Thus, hereafter, the commercial CE instrument was utilized.
As the AnchorChipTM plate could be damaged by the voltage applied during the off-line coupling, an AKD
plate was used. As described in chapter 3. Materials and Methods, multiple adjustments to the setup
were needed in order to maintain a stable current throughout the system. Another phenomenon that
occurred was that the CE separation worsen when moving the capillary outlet from inside the instrument,
as in stand alone CE, to the MALDI plate. Figure 32 presents the electropherogram of trypsin digested
human IgG, sample ④, obtained before coupling the instruments. This experiment was performed so
that the following electropherograms could be compared to this one and changes when coupling the
instruments could be detected. Also, as the CE software requires to be programmed prior to the run, it
was needed to know the retention time of the most relevant peaks.
The following step was to proceed with the off-line hyphenation of CE and MALDI-MS. Figure 33
illustrates the result of the first attempt to couple the instruments, the electropherogram obtained (top
panel) along with the respective current (bottom panel). The current was interrupted so that the user
could manually change the position of the capillary for the outlet vial attached to the outside of the CE
instrument to the robot arm. Two minutes were needed to change the position of the separation capillary.
Figure 32 - CE electropherogram of digested human IgG 1 mg/mL; injection: 20 kV
for 20 s; voltage: 25 kV; buffer: sodium phosphate (pH=3.2; 100 mM)
44
As explained previously, knowing the capillary length from the inlet to the UV-detector and from there to
the outlet, and assuming that the electrophoretic mobility is constant, it is possible to determine when
the analytes reach the MALDI plate. In the experiment of Figure 32, the capillary ⑭ was used (total
length: 87,5 cm; effective length: 24 cm).
At this moment, the goal was to detect the cluster of peaks with a retention time starting at 5 minutes.
Therefore, the deposition should be ready after around 14 minutes of separation. The voltage was
dropped to zero, the capillary was inserted in the robotic arm and a command was given in Arduino to
start the deposition. Note that experimental setup ① was used.
It is possible to observe that straight after turning the voltage back, the current suffered fluctuations,
then it got stable again and later dropped to zero in a step. No alteration was performed in the system
justificatory of this abrupt current drop. Resultant from this drop, the capillary broke close to the outlet.
The capillary ⑭ became too short to use mode ①, so the experiment setup was adapted to mode ②
of operation. Then a plastic piece with four orifices piece was used: one of them was covered and in the
others, capillaries were inserted. The separation capillary on the left, its remaining (the part that broke)
on the bottom and the capillary used to create a spray effect on the right. In this mode, in the beginning
of the experiment, the capillary is placed the MALDI plate.
Figure 34 illustrates the electropherogram obtained using experimental setup ② (top panel) along with
the respective current (bottom panel). Even with a stable current (first 7 minutes) and before starting the
deposition, that is, the capillaries were still on one MALDI plate spot; there were no peaks observed. It
was not expected that, only by changing the initial position of the separation capillary from the outlet vial
to the positioning table, the separation would be jeopardized. After 7 minutes of experiment, the current
suffers several fluctuations and latter ramped down to zero.
Figure 33 - CE electropherogram of digested human IgG 1 mg/mL from CE-MALDI
experimental setup ①; injection: 20 kV for 20 s; voltage: 25 kV; buffer: sodium
phosphate (pH=3.2; 100 mM)
45
Several more experiments were performed. One of three possible outcomes occurred: the capillary
broke during the run, no peaks were detected or too unstable current. That said, further setup
optimization is required in future works.
Even though a reproducible setup was not achieved, it could still be possible that the IgG sample was
reaching the MALDI plate. However, after analyzing the MALDI plate spots, it can be concluded that
either no IgG was present in the spots or the concentration was too low to be detected.
By analyzing digested IgG with MALDI-TOF-MS with no previous separation, as is the initial section of
the present chapter, but using the AKD target plate much lower intensities were obtained when
compared to the AnchorChipTM plate. Both DHB and HCCA matrices were used and similar poor results
were achieved. Later, it was verified that there were some difficulties encountered while the preparation
of AKD batch of that could also contribute to outcome of this investigation.
4.4. Comparison between ESI-MS and MALDI-TOF-MS
Overall, ESI and MALDI greatly improved biological mass spectrometry and particularly proteomics for
being soft ionization methods that can transform large biomolecules into ions. However, there are
numerous differences between the techniques.
MALDI results mainly in singly charged ions. In this project, this proved to be a large advantage as it
decreases the mass spectrum complexity when analyzing peptides. By opposition, ESI results in multiply
charged ions.
In MALDI much higher 𝑚/𝑧 were detected Though, this could be due to the higher charged that occurs
in ESI.
Figure 34 - CE electropherogram of digested human IgG 1 mg/mL from CE-MALDI
experimental setup ②; injection: 20 kV for 20 s; voltage: 25 kV; buffer: sodium
phosphate (pH=3.2; 100 mM)
[Atraia a atenção do seu leitor colocando uma boa citação no documento ou utilize
46
The steps before the analysis itself also vary. In ESI the molecules are ionized directly from the liquid
phase, the only preparation required is to mix the sample with the sheath liquid. As for MALDI, the
preparation step is much more time consuming. Waiting until the sample deposited in each spot is
completely dried to create the second layer of matrix, can take up to 30 minutes.
The hyphenation setup was clearly much simpler with ESI. The only thing to adapt was the CE cassette
and approximate the instrument as much as possible no decrease the required total capillary length.
However, introducing the capillary in the correct position of the ESI sprayer tip is not a straight forward
task. As for the off-line CE-MALDI-MS hyphenation, a setup had to be developed. In both cases, the
hyphenation was not successful. When coupling the separation with the first mass spectrometry
technique, it was noticed capillary coating was damaged, and therefore no further investigation was
possible. The ESI parameters need to be optimized for each analyte/setup. One possible justification
on why peaks were not being detected might be that the parameters were well not adjusted.
In opposition, in the hyphenation between CE and MALDI, the main issue was not in the second
instrument. In fact, no complete hyphenation was achieved. The setup was not fully developed and
therefore no conclusions can be taken from the hyphenation itself.
It is also important to refer that using ESI, it was not possible to identify the sample by performing a
database search. On the contrary, with MALDI IgG was not only identified, but the four types were
detected with very high scores. Hence, for this study, MALDI proved to be more effective.
47
5. Conclusions and Future Works
This final chapter introduces a summary of the main conclusions of the present project, as well as a
review of possible future investigations.
Before starting the analysis of IgG samples, an effective procedure to coat the capillary with PVA was
developed. The use of a specific tool that allowed a pressure injection was determinant in the procedure.
Several PVA concentrations were tested as well as methods to dissolve the PVA in water. The most
successful results were achieved using water soluble PVA (with a molecular mass of 30-70 kDa),
dissolved in cold water to achieve a concentration of 10% w/w. Throughout the development of a PVA
coating capillary method, it was noted that wetting the capillary with water before injecting the PVA,
decreased the probability of clogging.
In the first phase of the experimental work, a suitable method was developed for the separation of bovine
IgG through capillary electrophoresis. The best buffer proved to be sodium phosphate (pH=3.2;
concentration=100 mM).
A later step was to couple CE with MS. The first technique used was ESI. To do so, the buffer used in
the separation could not be a phosphoric buffer, as salts interfere with ESI. Therefore, the CE method
was optimized for the use of a buffer solution of ammonium acetate. The best results were obtained when
using a pH of 4.2 with a concentration of 25 mM, yet worse resolution and fewer peaks than with the
phosphoric buffer. To that extend, further optimization should be carried out to improve the outcome
when using this buffer.
Before coupling the instruments, tests with ESI stand alone were performed. Human IgG was analyzed,
yet no matches were detected when conducting a database search. Therefore, the experimental peak
list was compared with the masses of theoretical trypsin digested IgG and with the most common
contaminations in MS. Some peaks could possibly be from IgG, but no confirmation was possible given
the poor shape and resolution of the peaks. Additionally, triton and PEG contaminations were detected
and effectively suppressed.
The capillary was adapted to CE-ESI-MS and only then, hyphenation was put in place. In none of the
experiments done with this setup it was possible to detect peaks in ESI when expected based on the
retention times of CE and the capillary length (effective and total). Therefore, it is possible to conclude
that the hyphenation was not fully accomplished. Moreover, it was possible to observe peaks from
capillary coating in the mass spectrometer detector. As the capillary wall coating was damaged, the
investigation was not concluded. That said, future investigation could be focused on the optimization of
the ESI parameters, particularly when this instrument is coupled to the CE.
48
The decision was then made to try to another mass spectrometry technique: MALDI-TOF-MS. When
using this technique, excellent results were obtained. When performing a database search with the
results obtained, all four types of human IgG were a hit, with emphasis on IgG 1. Tandem MS was
carried out on the relevant peaks and again, when doing a database search the maximum score of
100% to IgG 1 was obtained.
Off-line coupling of CE and MALDI-TOF was not fully succeed in this project. Several capillaries broke
during the runs, but it was not possible to confirm the cause. Instabilities in the current could be related
with small fractures in the capillaries, however looking through a magnifying glass, fractures were not
detected. Due to these reasons it was not possible to conclude the runs. Therefore, a future work could
be to complement this study on the optimization of this setup with a detailed analysis on the feasibility
of using two different capillaries for the deposition onto the MALDI plate when there is no EOF. That is,
using one capillary for the separation itself and a second to obtain a spray effect and allow the analytes
deposition on the MALDI plate. In this project, the spray effect was accomplished using the BGE. Other
options should be investigated.
With the results from this project, it is possible to confirm that MALDI-TOF-MS is a powerful tool to
analyze peptides. However, one cannot conclude that ESI is not capable only because, in this specific
case, it could not identify the IgG peptides. It can simply be emphasized the complexity of implementing
a suitable and reliable experimental setup.
As CRPS is reportedly caused by mutations in the glycosylation of the IgG, when an effective
experimental setup is accomplished, glycosylation of IgG should be addressed. The final goal is to
investigate the glycosylation of IgG in blood samples from CRPS patients.
49
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53
A. Budapest Criteria for CRPS
Budapest clinical diagnostic criteria for CRPS [49]
(1) Continuing pain, which is disproportionate to any inciting event
(2) Must report at least one symptom in three of the four following categories:
• Sensory: reports of hyperesthesia and/or allodynia
• Vasomotor: reports of temperature asymmetry and/or skin color changes and/or skin color
asymmetry
• Sudomotor/edema: reports of edema and/or sweating changes and/or sweating asymmetry
• Motor/trophic: reports of decreased range of motion and/or motor dysfunction (weakness, tremor,
dystonia) and/or trophic changes (hair, nail, skin)
(3) Must display at least one sign at time of evaluation in two or more of the following categories:
• Sensory: evidence of hyperalgesia (to pinprick) and/or allodynia (to light touch and/or deep
somatic pressure and/or joint movement)
• Vasomotor: evidence of temperature asymmetry and/or skin color changes and/or asymmetry
• Sudomotor/edema: evidence of edema and/or sweating changes and/or sweating asymmetry
• Motor/trophic: evidence of decreased range of motion and/or motor dysfunction (weakness,
tremor, dystonia) and/or trophic changes (hair, nail, skin)
(4) There is no other diagnosis that better explains the signs and symptoms
54
B. List of Digested Peptides from IgG
The tables presented in this chapter include the list of trypsin digested peptides from human IgG, types 1 to 4, obtained through reference [50]. A maximum of
three miss-cleavages (MC) was allowed. In column MC the number of miss-cleavages is shown (from 0 to 3).
Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 - 330
Mass Position MC Peptide Sequence
9456.7722 1-93 3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTK
9411.7507 5-96 3 GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDK
9069.5604 5-93 2 GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTK
8372.2169 17-97 3 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKK
8244.1219 17-96 2 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDK
7901.9316 17-93 1 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTK
7579.8369 31-101 3 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
7268.6031 106-171 3 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
7126.5782 31-97 2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
6998.4832 31-96 1 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
6656.2929 31-93 0 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
6070.9218 239-292 3 DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
6042.9821 102-157 3 SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
6040.9476 244-297 3 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
5609.8190 106-157 2 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
5484.6256 244-292 2 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
5483.6739 228-275 3 EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
5398.6048 276-322 3 TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK
5393.5807 139-184 3 TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
5198.4905 254-299 3 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
5119.5812 158-200 3 FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
5039.5029 132-175 3 DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
55
Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 – 330 (continued)
Mass Position MC Peptide Sequence
4955.3573 254-297 2 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
4557.2064 132-171 2 DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
4433.2527 98-138 3 VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
4399.0353 254-292 1 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
4313.1342 293-330 3 LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
4223.0865 139-175 2 TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
4216.0179 239-275 2 DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
3979.9940 102-138 2 SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
3880.9980 172-203 3 TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
3756.8122 298-330 2 SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
3744.9313 97-131 3 KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
3740.7900 139-171 1 TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
3629.8056 176-205 3 EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
3629.7216 244-275 1 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
3616.8364 98-131 2 VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
3546.8309 106-138 1 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
3543.7008 293-322 2 LTVDKSRWQQGNVFSCSVMHEALHNHYTQK
3513.6790 300-330 1 WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
3460.7971 172-200 2 TKPREEQYNSTYRVVSVLTVLHQDWLNGK
3398.7015 176-203 2 EEQYNSTYRVVSVLTVLHQDWLNGKEYK
3396.7943 224-253 3 GQPREPQVYTLPPSRDELTKNQVSLTCLVK
3330.5926 158-184 2 FNWYVDGVEVHNAKTKPREEQYNSTYR
3163.5777 102-131 1 SCDKTHTCPPCPAPELLGGPSVFLFPPKPK
2987.3788 298-322 1 SRWQQGNVFSCSVMHEALHNHYTQK
2978.5006 176-200 1 EEQYNSTYRVVSVLTVLHQDWLNGK
2958.5604 228-253 2 EPQVYTLPPSRDELTKNQVSLTCLVK
2898.4223 132-157 1 DTLMISRTPEVTCVVVDVSHEDPEVK
2887.5498 185-209 3 VVSVLTVLHQDWLNGKEYKCKVSNK
2819.4971 1-30 2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVK
2744.2456 300-322 0 WQQGNVFSCSVMHEALHNHYTQK
2730.4145 106-131 0 THTCPPCPAPELLGGPSVFLFPPKPK
2673.3770 276-299 2 TTPPVLDSDGSFFLYSKLTVDKSR
2544.1313 254-275 0 GFYPSDIAVEWESNGQPENNYK
2510.3361 222-243 3 AKGQPREPQVYTLPPSRDELTK
2459.3115 185-205 2 VVSVLTVLHQDWLNGKEYKCK
56
Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 – 330 (continued)
Peptide Sequence
Position MC Peptide Sequence
2432.2853 5-30 1 GPSVFPLAPSSKSTSGGTAALGCLVK
2430.2438 276-297 1 TTPPVLDSDGSFFLYSKLTVDK
2353.2986 218-238 3 TISKAKGQPREPQVYTLPPSR
2311.2040 224-243 2 GQPREPQVYTLPPSRDELTK
2228.2073 185-203 1 VVSVLTVLHQDWLNGKEYK
2160.0984 158-175 1 FNWYVDGVEVHNAKTKPR
2082.0059 139-157 0 TPEVTCVVVDVSHEDPEVK
1927.1044 204-221 3 CKVSNKALPAPIEKTISK
1924.0399 222-238 2 AKGQPREPQVYTLPPSR
1918.0466 201-217 3 EYKCKVSNKALPAPIEK
1905.1279 210-227 3 ALPAPIEKTISKAKGQPR
1895.1324 206-223 3 VSNKALPAPIEKTISKAK
1873.9218 276-292 0 TTPPVLDSDGSFFLYSK
1872.9701 228-243 1 EPQVYTLPPSRDELTK
1808.0064 185-200 0 VVSVLTVLHQDWLNGK
1724.9078 224-238 1 GQPREPQVYTLPPSR
1696.0003 206-221 2 VSNKALPAPIEKTISK
1690.9044 239-253 1 DELTKNQVSLTCLVK
1677.8019 158-171 0 FNWYVDGVEVHNAK
1671.8085 172-184 1 TKPREEQYNSTYR
1573.8584 1-16 1 ASTKGPSVFPLAPSSK
1497.8457 204-217 2 CKVSNKALPAPIEK
1466.8940 210-223 2 ALPAPIEKTISKAK
1375.7249 94-105 3 VDKKVEPKSCDK
1286.6739 228-238 0 EPQVYTLPPSR
1267.7620 210-221 1 ALPAPIEKTISK
1266.7416 206-217 1 VSNKALPAPIEK
1264.6565 17-30 0 STSGGTAALGCLVK
1189.5120 176-184 0 EEQYNSTYR
1186.6466 5-16 0 GPSVFPLAPSSK
1104.6081 244-253 0 NQVSLTCLVK
1098.5612 201-209 2 EYKCKVSNK
1085.6425 218-227 2 TISKAKGQPR
1033.5346 97-105 2 KVEPKSCDK
57
Table 7 - Chain Immunoglobulin heavy constant gamma 1 at positions <1 – 330 (continued
Position MC Position Peptide Sequence
942.5618 94-101 2 VDKKVEPK
905.4397 98-105 1 VEPKSCDK
838.5032 210-217 0 ALPAPIEK
835.4342 132-138 0 DTLMISR
818.4730 293-299 1 LTVDKSR
788.4512 323-330 0 SLSLSPGK
678.3603 204-209 1 CKVSNK
670.3229 201-205 1 EYKCK
656.3838 222-227 1 AKGQPR
647.4086 218-223 1 TISKAK
605.3141 239-243 0 DELTK
600.3715 97-101 1 KVEPK
575.3399 293-297 0 LTVDK
501.3143 172-175 0 TKPR
489.3031 94-97 1 VDKK
472.2765 98-101 0 VEPK
457.2517 224-227 0 GQPR
452.1809 102-105 0 SCDK
448.2766 218-221 0 TISK
447.2562 206-209 0 VSNK
439.2187 201-203 0 EYK
406.2296 1-4 0 ASTK
361.2081 94-96 0 VDK
262.1510 298-299 0 SR
250.1220 204-205 0 CK
218.1499 222-223 0 AK
147.1128 97-97 0 K
58
Table 8 - Chain Immunoglobulin heavy constant gamma 2 at positions <1-326
Mass Position MC Peptide Sequence
9652.7301 1-93 3 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSNFGTQTYTCNVDHKPSNTK
9607.7086 5-96 3 GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSN FGTQTYTCNVDHKPSNTKVDK
9265.5183 5-93 2 GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSN FGTQTYTCNVDHKPSNTK
8881.3563 17-100 3 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDH KPSNTKVDKTVER
8396.0965 17-96 2 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDH KPSNTKVDK
8053.9062 17-93 1 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDH KPSNTK
7700.7644 102-171 3 CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR
7661.7808 31-101 3 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK
7533.6859 31-100 2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER
7346.5629 101-167 3 KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK
7218.4679 102-167 2 CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK
7136.5274 135-196 3 TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
7048.4261 31-96 1 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK
6706.2358 31-93 0 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK
6177.9708 128-180 3 DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR
6150.8609 235-288 3 EEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSK
6088.9146 240-293 3 NQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK
5532.5926 240-288 2 NQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSK
5531.6409 224-271 3 EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYK
5430.5768 272-318 3 TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK
5361.5545 135-180 2 TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR
5246.4575 250-295 3 GFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR
5039.4665 128-171 2 DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR
5003.3243 250-293 2 GFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK
4557.1700 128-167 1 DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK
4447.0023 250-288 1 GFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSK
4313.1342 289-326 3 LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
4263.9849 235-271 2 EEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYK
4223.0501 135-171 1 TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR
59
Table 8 - Chain Immunoglobulin heavy constant gamma 2 at positions <1-326 (continued)
Mass Position MC Peptide Sequence
4110.0869 97-134 3 TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR
3834.9925 168-199 3 TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK
3756.8122 294-326 2 SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
3740.7536 135-167 0 TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK
3645.7166 240-271 1 NQVSLTCLVKGFYPSDISVEWESNGQPENNYK
3635.8608 94-127 3 VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPK
3624.8271 101-134 2 KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR
3583.8002 172-201 3 EEQFNSTFRVVSVLTVVHQDWLNGKEYKCK
3543.7008 289-318 2 LTVDKSRWQQGNVFSCSVMHEALHNHYTQK
3513.6790 296-326 1 WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
3496.7321 102-134 1 CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR
3428.7664 220-249 3 GQPREPQVYTLPPSREEMTKNQVSLTCLVK
3414.7916 168-196 2 TKPREEQFNSTFRVVSVLTVVHQDWLNGK
3352.6960 172-199 2 EEQFNSTFRVVSVLTVVHQDWLNGKEYK
3293.6705 97-127 2 TVERKCCVECPPCPAPPVAGPSVFLFPPKPK
2990.5325 224-249 2 EPQVYTLPPSREEMTKNQVSLTCLVK
2987.3788 294-318 1 SRWQQGNVFSCSVMHEALHNHYTQK
2965.5121 1-30 2 ASTKGPSVFPLAPCSRSTSESTAALGCLVK
2932.4951 172-196 1 EEQFNSTFRVVSVLTVVHQDWLNGK
2873.5342 181-205 3 VVSVLTVVHQDWLNGKEYKCKVSNK
2808.4107 101-127 1 KCCVECPPCPAPPVAGPSVFLFPPKPK
2744.2456 296-318 0 WQQGNVFSCSVMHEALHNHYTQK
2705.3490 272-295 2 TTPPMLDSDGSFFLYSKLTVDKSR
2680.3158 102-127 0 CCVECPPCPAPPVAGPSVFLFPPKPK
2578.3003 5-30 1 GPSVFPLAPCSRSTSESTAALGCLVK
2572.3188 218-239 3 TKGQPREPQVYTLPPSREEMTK
2560.1262 250-271 0 GFYPSDISVEWESNGQPENNYK
2462.2159 272-293 1 TTPPMLDSDGSFFLYSKLTVDK
2445.2958 181-201 2 VVSVLTVVHQDWLNGKEYKCK
2383.3092 214-234 3 TISKTKGQPREPQVYTLPPSR
2343.1761 220-239 2 GQPREPQVYTLPPSREEMTK
2214.1917 181-199 1 VVSVLTVVHQDWLNGKEYK
1954.0504 218-234 2 TKGQPREPQVYTLPPSR
1921.1229 206-223 3 GLPAPIEKTISKTKGQPR
60
Table 8 - Chain Immunoglobulin heavy constant gamma 2 at positions <1-326 (continued)
Mass Position MC Peptide Sequence
1913.0888 200-217 3 CKVSNKGLPAPIEKTISK
1911.1273 202-219 3 VSNKGLPAPIEKTISKTK
1905.8939 272-288 0 TTPPMLDSDGSFFLYSK
1904.9422 224-239 1 EPQVYTLPPSREEMTK
1904.0309 197-213 3 EYKCKVSNKGLPAPIEK
1793.9908 181-196 0 VVSVLTVVHQDWLNGK
1724.9078 220-234 1 GQPREPQVYTLPPSR
1722.8764 235-249 1 EEMTKNQVSLTCLVK
1681.9846 202-217 2 VSNKGLPAPIEKTISK
1639.8187 168-180 1 TKPREEQFNSTFR
1617.8417 1-16 1 ASTKGPSVFPLAPCSR
1483.8301 200-213 2 CKVSNKGLPAPIEK
1482.8890 206-219 2 GLPAPIEKTISKTK
1366.6882 17-30 0 STSESTAALGCLVK
1286.6739 224-234 0 EPQVYTLPPSR
1253.7463 206-217 1 GLPAPIEKTISK
1252.7259 202-213 1 VSNKGLPAPIEK
1230.6299 5-16 0 GPSVFPLAPCSR
1157.5222 172-180 0 EEQFNSTFR
1115.6531 214-223 2 TISKTKGQPR
1104.6081 240-249 0 NQVSLTCLVK
1098.5612 197-205 2 EYKCKVSNK
974.5629 94-101 2 VDKTVERK
846.4679 94-100 1 VDKTVER
835.4342 128-134 0 DTLMISR
824.4876 206-213 0 GLPAPIEK
818.4730 289-295 1 LTVDKSR
788.4512 319-326 0 SLSLSPGK
686.3944 218-223 1 TKGQPR
678.3603 200-205 1 CKVSNK
677.4192 214-219 1 TISKTK
670.3229 197-201 1 EYKCK
637.2861 235-239 0 EEMTK
632.3726 97-101 1 TVERK
61
Table 8 - Chain Immunoglobulin heavy constant gamma 2 at positions <1-326 (continued)
Mass Position MC Peptide Sequence
575.3399 289-293 0 LTVDK
504.2776 97-100 0 TVER
501.3143 168-171 0 TKPR
457.2517 220-223 0 GQPR
448.2766 214-217 0 TISK
447.2562 202-205 0 VSNK
439.2187 197-199 0 EYK
406.2296 1-4 0 ASTK
361.2081 94-96 0 VDK
262.1510 294-295 0 SR
250.1220 200-201 0 CK
248.1605 218-219 0 TK
147.1128 101-101 0 K
62
Table 9 - Immunoglobulin heavy constant gamma 3 at positions 1-377
Mass Position MC Peptide Sequence
9488.7191 1-93 3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYTCNVNHKPSNTK
9443.6976 5-96 3 GPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYTCNVNHKPSNTKVDK
9101.5073 5-93 2 GPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYTCNVNHKPSNTK
8388.1866 17-97 3 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNH KPSNTKVDKR
8232.0855 17-96 2 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNH KPSNTKVDK
7889.8952 17-93 1 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNH KPSNTK
7611.8380 31-101 3 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELK
7427.4207 301-365 3 GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNR
7361.4591 275-339 3 EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSK
7142.5479 31-97 2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKR
6986.4468 31-96 1 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDK
6650.1251 286-344 3 EEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDK
6646.3498 159-218 3 CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAK
6644.2565 31-93 0 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTK
6302.1142 149-206 3 SCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFK
6274.9899 291-346 3 NQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSR
6093.8030 286-339 2 EEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSK
6031.8568 291-344 2 NQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDK
5475.5347 291-339 1 NQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSK
5391.6014 186-231 3 TPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFR
5248.6770 159-206 2 CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFK
5189.3996 301-346 2 GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSR
5053.5185 179-222 3 DTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPR
4946.2665 301-344 1 GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDK
4842.4750 207-247 3 WYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGK
4755.2722 134-178 3 SCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPK
4571.2220 179-218 2 DTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAK
4518.2514 144-185 3 CPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISR
4389.9444 301-339 0 GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSK
4237.1022 186-222 2 TPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPR
3963.9991 149-185 2 SCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISR
3865.0031 219-250 3 TKPREEQYNSTFRVVSVLTVLHQDWLNGKEYK
63
Table 9 - Immunoglobulin heavy constant gamma 3 at positions 1-377 (continued)
Mass Position MC Peptide Sequence
3773.8751 345-377 3 SRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
3754.8057 186-218 1 TPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAK
3701.8350 144-178 2 CPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPK
3613.8107 223-252 3 EEQYNSTFRVVSVLTVLHQDWLNGKEYKCK
3560.7637 340-369 3 LTVDKSRWQQGNIFSCSVMHEALHNRFTQK
3530.7419 347-377 2 WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
3460.5574 102-133 3 TPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPK
3444.8022 219-247 2 TKPREEQYNSTFRVVSVLTVLHQDWLNGK
3428.7664 271-300 3 GQPREPQVYTLPPSREEMTKNQVSLTCLVK
3382.7066 223-250 2 EEQYNSTFRVVSVLTVLHQDWLNGKEYK
3375.5952 98-128 3 VELKTPLGDTTHTCPRCPEPKSCDTPPPCPR
3234.3967 114-143 3 CPEPKSCDTPPPCPRCPEPKSCDTPPPCPR
3234.3967 119-148 3 SCDTPPPCPRCPEPKSCDTPPPCPRCPEPK
3234.3967 129-158 3 CPEPKSCDTPPPCPRCPEPKSCDTPPPCPR
3173.5493 179-206 1 DTLMISRTPEVTCVVVDVSHEDPEVQFK
3147.5827 149-178 1 SCDTPPPCPRCPAPELLGGPSVFLFPPKPK
3056.4941 340-365 2 LTVDKSRWQQGNIFSCSVMHEALHNR
3053.4864 207-231 2 WYVDGVEVHNAKTKPREEQYNSTFR
3004.4417 345-369 2 SRWQQGNIFSCSVMHEALHNRFTQK
2990.5325 275-300 2 EPQVYTLPPSREEMTKNQVSLTCLVK
2962.5057 223-247 1 EEQYNSTFRVVSVLTVLHQDWLNGK
2910.5619 159-185 1 CPAPELLGGPSVFLFPPKPKDTLMISR
2906.3052 102-128 2 TPLGDTTHTCPRCPEPKSCDTPPPCPR
2887.5498 232-256 3 VVSVLTVLHQDWLNGKEYKCKVSNK
2863.4804 1-30 2 ASTKGPSVFPLAPCSRSTSGGTAALGCLVK
2761.3086 347-369 1 WQQGNIFSCSVMHEALHNRFTQK
2680.1444 119-143 2 SCDTPPPCPRCPEPKSCDTPPPCPR
2680.1444 134-158 2 SCDTPPPCPRCPEPKSCDTPPPCPR
2572.3188 269-290 3 TKGQPREPQVYTLPPSREEMTK
2500.1721 345-365 1 SRWQQGNIFSCSVMHEALHNR
2478.2591 97-118 3 RVELKTPLGDTTHTCPRCPEPK
2476.2686 5-30 1 GPSVFPLAPCSRSTSGGTAALGCLVK
2459.3115 232-252 2 VVSVLTVLHQDWLNGKEYKCK
2383.3092 265-285 3 TISKTKGQPREPQVYTLPPSR
2357.1329 186-206 0 TPEVTCVVVDVSHEDPEVQFK
2343.1761 271-290 2 GQPREPQVYTLPPSREEMTK
2322.1580 98-118 2 VELKTPLGDTTHTCPRCPEPK
2266.1972 94-113 3 VDKRVELKTPLGDTTHTCPR
64
Table 9 - Immunoglobulin heavy constant gamma 3 at positions 1-377 (continued)
Mass Position MC Peptide Sequence
2257.0389 347-365 0 WQQGNIFSCSVMHEALHNR
2228.2073 232-250 1 VVSVLTVLHQDWLNGKEYK
2180.9595 114-133 2 CPEPKSCDTPPPCPRCPEPK
2180.9595 129-148 2 CPEPKSCDTPPPCPRCPEPK
2094.1456 159-178 0 CPAPELLGGPSVFLFPPKPK
1954.0504 269-285 2 TKGQPREPQVYTLPPSR
1935.1385 257-274 3 ALPAPIEKTISKTKGQPR
1927.1044 251-268 3 CKVSNKALPAPIEKTISK
1925.1429 253-270 3 VSNKALPAPIEKTISKTK
1924.0069 97-113 2 RVELKTPLGDTTHTCPR
1918.0466 248-264 3 EYKCKVSNKALPAPIEK
1904.9422 275-290 1 EPQVYTLPPSREEMTK
1898.9871 207-222 1 WYVDGVEVHNAKTKPR
1852.8680 102-118 1 TPLGDTTHTCPRCPEPK
1808.0064 232-247 0 VVSVLTVLHQDWLNGK
1767.9058 98-113 1 VELKTPLGDTTHTCPR
1724.9078 271-285 1 GQPREPQVYTLPPSR
1722.8764 286-300 1 EEMTKNQVSLTCLVK
1696.0003 253-268 2 VSNKALPAPIEKTISK
1655.8136 219-231 1 TKPREEQYNSTFR
1626.7073 114-128 1 CPEPKSCDTPPPCPR
1626.7073 119-133 1 SCDTPPPCPRCPEPK
1626.7073 129-143 1 CPEPKSCDTPPPCPR
1626.7073 134-148 1 SCDTPPPCPRCPEPK
1626.7073 144-158 1 CPEPKSCDTPPPCPR
1617.8417 1-16 1 ASTKGPSVFPLAPCSR
1497.8457 251-264 2 CKVSNKALPAPIEK
1496.9046 257-270 2 ALPAPIEKTISKTK
1416.6906 207-218 0 WYVDGVEVHNAK
1298.6157 102-113 0 TPLGDTTHTCPR
1292.7208 366-377 1 FTQKSLSLSPGK
1286.6739 275-285 0 EPQVYTLPPSR
1267.7620 257-268 1 ALPAPIEKTISK
1266.7416 253-264 1 VSNKALPAPIEK
1264.6565 17-30 0 STSGGTAALGCLVK
1230.6299 5-16 0 GPSVFPLAPCSR
1173.5171 223-231 0 EEQYNSTFR
1115.6531 265-274 2 TISKTKGQPR
65
Table 9 - Immunoglobulin heavy constant gamma 3 at positions 1-377 (continued)
Mass Position MC Peptide Sequence
1104.6081 291-300 0 NQVSLTCLVK
1098.5612 248-256 2 EYKCKVSNK
1072.4550 119-128 0 SCDTPPPCPR
1072.4550 134-143 0 SCDTPPPCPR
1072.4550 149-158 0 SCDTPPPCPR
986.5993 94-101 2 VDKRVELK
838.5032 257-264 0 ALPAPIEK
835.4342 179-185 0 DTLMISR
818.4730 340-346 1 LTVDKSR
788.4512 370-377 0 SLSLSPGK
686.3944 269-274 1 TKGQPR
678.3603 251-256 1 CKVSNK
677.4192 265-270 1 TISKTK
670.3229 248-252 1 EYKCK
644.4090 97-101 1 RVELK
637.2861 286-290 0 EEMTK
575.3399 340-344 0 LTVDK
573.2701 114-118 0 CPEPK
573.2701 129-133 0 CPEPK
573.2701 144-148 0 CPEPK
523.2875 366-369 0 FTQK
517.3092 94-97 1 VDKR
501.3143 219-222 0 TKPR
488.3078 98-101 0 VELK
457.2517 271-274 0 GQPR
448.2766 265-268 0 TISK
447.2562 253-256 0 VSNK
439.2187 248-250 0 EYK
406.2296 1-4 0 ASTK
361.2081 94-96 0 VDK
262.1510 345-346 0 SR
250.1220 251-252 0 CK
248.1605 269-270 0 TK
175.1189 97-97 0 R
66
Table 10 -– Immunoglobulin heavy constant gamma 4 at positions <1-327
Mass Position MC Peptide Sequence
9204.5594 5-93 3 GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTK
8335.1376 17-96 3 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDK
8003.0408 1-79 3 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTK
7992.9473 17-93 2 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTK
7840.8626 102-172 3 YGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
7801.8040 98-168 3 VESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK
7615.8290 5-79 2 GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTK
7370.5136 225-289 3 EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
7358.5660 102-168 2 YGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK
7157.5377 136-197 3 TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK
7143.5683 31-97 3 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR
6987.4672 31-96 2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK
6645.2769 31-93 1 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK
6404.2169 17-79 1 STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK
6184.9654 129-181 3 DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR
6068.9538 241-294 3 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK
5925.8374 221-272 3 GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
5512.6317 241-289 2 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
5487.6035 225-272 2 EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
5427.5949 273-319 3 TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK
5368.5491 136-181 2 TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR
5226.4966 251-296 3 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR
5056.5465 31-79 0 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK
5030.4662 129-172 2 DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
4983.3635 251-294 2 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK
4548.1697 129-168 1 DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK
4427.0414 251-289 1 GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
4330.1495 290-327 3 LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
4245.1697 97-135 3 RVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISR
4214.0498 136-172 1 TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
4089.0686 98-135 2 VESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISR
3865.0031 169-200 3 TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
3773.8275 295-327 2 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
67
Table 10 -– Immunoglobulin heavy constant gamma 4 at positions <1-327 (continued)
Mass Position MC Peptide Sequence
3770.9436 94-128 3 VDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPK
3731.7533 136-168 0 TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK
3645.8306 102-135 1 YGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISR
3629.7216 241-272 1 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
3613.8107 173-202 3 EEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
3599.8560 219-250 3 AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
3544.6848 290-319 2 LTVDKSRWQEGNVFSCSVMHEALHNHYTQK
3530.6943 297-327 1 WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
3444.8022 169-197 2 TKPREEQFNSTYRVVSVLTVLHQDWLNGK
3428.7533 97-128 2 RVESKYGPPCPSCPAPEFLGGPSVFLFPPKPK
3400.7239 221-250 2 GQPREPQVYTLPPSQEEMTKNQVSLTCLVK
3382.7066 173-200 2 EEQFNSTYRVVSVLTVLHQDWLNGKEYK
3272.6522 98-128 1 VESKYGPPCPSCPAPEFLGGPSVFLFPPKPK
2988.3628 295-319 1 SRWQEGNVFSCSVMHEALHNHYTQK
2965.5121 1-30 2 ASTKGPSVFPLAPCSRSTSESTAALGCLVK
2962.5057 173-197 1 EEQFNSTYRVVSVLTVLHQDWLNGK
2962.4900 225-250 1 EPQVYTLPPSQEEMTKNQVSLTCLVK
2943.5244 215-240 3 TISKAKGQPREPQVYTLPPSQEEMTK
2887.5498 182-206 3 VVSVLTVLHQDWLNGKEYKCKVSNK
2829.4142 102-128 0 YGPPCPSCPAPEFLGGPSVFLFPPKPK
2745.2296 297-319 0 WQEGNVFSCSVMHEALHNHYTQK
2701.3831 273-296 2 TTPPVLDSDGSFFLYSRLTVDKSR
2578.3003 5-30 1 GPSVFPLAPCSRSTSESTAALGCLVK
2549.2776 80-101 3 TYTCNVDHKPSNTKVDKRVESK
2544.1313 251-272 0 GFYPSDIAVEWESNGQPENNYK
2514.2657 219-240 2 AKGQPREPQVYTLPPSQEEMTK
2459.3115 182-202 2 VVSVLTVLHQDWLNGKEYKCK
2458.2500 273-294 1 TTPPVLDSDGSFFLYSRLTVDK
2315.1336 221-240 1 GQPREPQVYTLPPSQEEMTK
2228.2073 182-200 1 VVSVLTVLHQDWLNGKEYK
2106.0396 80-97 2 TYTCNVDHKPSNTKVDKR
1949.9385 80-96 1 TYTCNVDHKPSNTKVDK
1919.0630 201-218 3 CKVSNKGLPSSIEKTISK
1910.0051 198-214 3 EYKCKVSNKGLPSSIEK
1901.9279 273-289 0 TTPPVLDSDGSFFLYSR
1897.0865 207-224 3 GLPSSIEKTISKAKGQPR
1887.0909 203-220 3 VSNKGLPSSIEKTISKAK
1876.8997 225-240 0 EPQVYTLPPSQEEMTK
1808.0064 182-197 0 VVSVLTVLHQDWLNGK
68
Table 10 -– Immunoglobulin heavy constant gamma 4 at positions <1-327 (continued)
Mass Position MC Peptide Sequence
1687.9588 203-218 2 VSNKGLPSSIEKTISK
1655.8136 169-181 1 TKPREEQFNSTYR
1617.8417 1-16 1 ASTKGPSVFPLAPCSR
1607.7482 80-93 0 TYTCNVDHKPSNTK
1489.8043 201-214 2 CKVSNKGLPSSIEK
1458.8526 207-220 2 GLPSSIEKTISKAK
1366.6882 17-30 0 STSESTAALGCLVK
1259.7205 207-218 1 GLPSSIEKTISK
1258.7001 203-214 1 VSNKGLPSSIEK
1230.6299 5-16 0 GPSVFPLAPCSR
1173.5171 173-181 0 EEQFNSTYR
1104.6081 241-250 0 NQVSLTCLVK
1098.5612 198-206 2 EYKCKVSNK
1085.6425 215-224 2 TISKAKGQPR
960.5472 94-101 2 VDKRVESK
835.4342 129-135 0 DTLMISR
830.4618 207-214 0 GLPSSIEK
818.4730 290-296 1 LTVDKSR
804.4825 320-327 0 SLSLSLGK
678.3603 201-206 1 CKVSNK
670.3229 198-202 1 EYKCK
656.3838 219-224 1 AKGQPR
647.4086 215-220 1 TISKAK
618.3569 97-101 1 RVESK
575.3399 290-294 0 LTVDK
517.3092 94-97 1 VDKR
501.3143 169-172 0 TKPR
462.2558 98-101 VESK
457.2517 221-224 0 GQPR
448.2766 215-218 0 TISK
447.2562 203-206 0 VSNK
439.2187 198-200 0 EYK
406.2296 1-4 0 ASTK
361.2081 94-96 0 VDK
262.1510 295-296 0 SR
250.1220 201-202 0 CK
218.1499 219-220 0 AK
175.1189 97-97 0 R
69
C. Biotools Analysis
Figure 35 – Results obtained from Biotools database search for MS analysis of IgG 1
(database: Swiss-Prot)
Figure 36 – Results obtained from Biotools database search for MS analysis of IgG 2
(database: Swiss-Prot)
70
Figure 37 – Results obtained from Biotools database search for MS analysis of IgG 3
(database: Swiss-Prot)
Figure 38 - Results obtained from Biotools database search for MS analysis of IgG 4
(database: Swiss-Prot)
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