Presented by

Upasana Ganguly

Proteomics is the large scale analysis ofproteins, particularly their structure andfunction.

Discovery of 2-D gels was a landmark inproteomics which provided the first feasibleway to display thousands of protein spots ona single gel.

In 1990s biological mass spectrometry haddeveloped into a sensitive and robusttechnique which gave proteomics its impetus.

Mass spectroscopy or Mass spectrometry(MS) is an instrumental approach that allowsthe mass measurement of ions derived frommolecules.

Mass spectrometers are capable of forming,separating, detecting molecular ions basedon their mass to charge ratio (m/z).

Among the well known analyticaltechniques, MS holds a special place as itmeasures an intrinsic property of amolecule – its mass.

MS has replaced Edman degradation.

Uses of MS in proteomics are in three majorareas

1. Characterization and quality control ofrecombinant proteins

2. Protein identification

3. Detection and characterization of post-translational modifications.

Ionization source, analyzer, detector areunder high vacuum to allow unhinderedmovement of ions

aspirin

Relative Abundance

120 m/z-for singly charged ion this is the mass

1897 – J.J Thomson constructed the first MassSpectrometer called „parabola spectrograph‟. Hewas awarded Noble prize in 1906.

1918-1919 – More sophisticated Massspectrometers by Arthur Dempster and FrancisAston.

1946 – W.F Stephens proposed the concept of Timeof Flight (TOF)

1950 – Wolfgang Paul developed Quadrupole massanalyzer and later Quadrupole ion trap massanalyzer.

1983 – First Ion trap became availablecommercially.

1988 – Soft Ionization techniques were introduced :MALDI and ESI.

Electron Impact (EI - Hard method)◦ small molecules, 1-1000 Daltons

Fast Atom Bombardment (FAB – Semi-hard)◦ peptides, sugars, up to 6000 Daltons

Electrospray Ionization (ESI - Soft)◦ peptides, proteins, up to 70,000 Daltons

Matrix Assisted Laser Desorption (MALDI-Soft)◦ peptides, proteins, DNA, up to 300 kD

MALDI first introduced in 1985 by FranzHillenkamp and Michael Karas (Frankfurt)

Mechanism :Analyte molecules are embedded in a bed of

specific wavelength (UV337nm) absorbing matrix

Dried to produce a co-crystallized mixture

Bombarded with short duration (1-10ns) pulses of UV light from a nitrogen laser

Ionization of both matrix and analyte molecules via an energy transfer mechanism from matrix to the embedded analyte

A high potential electric field is applied between the sample probe and orifice

Accelerates the ions to the mass analyzer

hn

Laser

UV 337nm

1. Sample (A) is mixed withexcess matrix (M) and driedon a MALDI plate.

2. Laser flash ionizes matrixmolecules.

3. Sample molecules areionized by proton transferfrom matrix:

MH+ + A M + AH+.

AH+

+30 kV

Variable

Ground

Grid Grid

Sample plate

Practical mass limit : ~300,000 Daltons

Sensitivity : low femtomole to low picomole

No fragmentation

Suitable for analysis of complex mixtures

Samples are added directly to the matrix

Precise ionization process : not known

Signal intensities : depend on incorporationof peptides into crystals

Masses below 500 Daltons are obscured bymatrix related ions

Low resolution

Background interference

Possibility of photodegradation by laserdesorption/ionization

Poor for determining peptide modifications

Electro-spray ionization first conceived in 1960‟sby Malcolm Dole but put into practice in 1980‟s byJohn Fenn (Yale).

Unique feature : both singly and multiplecharged ions can be formed.

Mechanism :A solution is introduced through a small diameter needle in the

presence of a strong electric field(3-5kV)

A fine spray of charged droplets is created

Charged droplets are desolvated by the application of a countercurrent flow of gas and/or heat causing the droplet to

evaporate

Explosion of droplet and expulsion of gas phase ions

Positive ion mode measures (M + H)+

If the sample has functional groups thatreadily accept H+ (such as amide andamino groups found in peptides andproteins) then positive ion detection isused-PROTEINS

Negative ion mode measures (M - H)-

If a sample has functional groups thatreadily lose a proton (such as carboxylicacids and hydroxyls as found in nucleicacids and sugars) then negative iondetection is used-DNA

Practical mass limit : 70,000 Daltons

Sensitivity : good

Can be easily coupled to LiquidChromatography (LC)

No matrix interference

Multiple charging gives better mass accuracy

Excellent for determining peptidemodifications

Low salt tolerance

Low tolerance for mixtures

Difficulty in cleaning overly contaminatedinstrument

Resolution is defined as the ability to

separate and measure the masses of ions

of similar but not identical molecular mass.

The better the resolution, the better the

instrument and the better the mass

accuracy.

Resolution is represented as:M

DM

Full Width Half Maximum 10% Valley

Mass

analyzers

Mass

separation

Time of Flight

(TOF)

Quadrupole

MS

Ion trap / FT

ICR

Structural

analysis

Tandem Mass

Spectrometry

or MS/MS

Measures m/z values of analytes by pulsingmolecular ions from the ionization sourceinto flight tubes.

All ions are accelerated across the samedistance by the same force, they same sameK.E. As velocity is dependent on K.E, lighterions travel faster.

m/z values are calculated by the time delayfrom formation of ion till it reaches to strikethe detector (Time of flight).

Linear Time Of Flight tube

Reflector Time Of Flight tube

detector

reflector

ion source

ion source

detector

time of flight

time of flight

Peaks are inherently broad in MALDI-TOFspectra (poor mass resolution).

Cause : Ions of the same mass coming fromthe target have different speeds. This is dueto uneven energy- distribution when theions are formed by the laser pulse.

Can be compensated with the use ofreflector TOF analyzer : a single stagegridded ion mirror that subjects the ions toa uniform repulsive electric field to reflectthem.

A quadrupole mass filter consists of fourparallel metal rods with different charges.

Two opposite rods have an applied +potential and the other two rods have a –potential.

The applied voltages affect the trajectory ofions traveling down the flight path.

For given dc and ac voltages, only ions of acertain mass-to-charge ratio pass throughthe quadrupole filter and all other ions arethrown out of their original path.

Represents a 3-D quadrupole mass analyzer.Consists of a ring electrode and two end-cap

electrodes. Small holes in end-cap electrodes allow

passage of ions into and out of the trap. RF voltage is applied to ring electrode while

end cap electrodes are held at ground.Oscillating potential difference between ring

and end cap electrodes forms a quadrupolarfield.

Similar to Quadrupole Ion trap MS. Reactions are carried out in a cell bound by

electrodes (trapping,excite and detect plates). Uses powerful magnet (5-10 Tesla) to create a

miniature cyclotron. m/z value is directly related to its cyclotron

frequency.

Use of Tandem Mass Spectroscopy is to inducefragmentation and obtain structural information.

Series of events:

1. Mass selection of a precursor ion

2. Intermediate reaction event

3. Analysis of product ions or fragment ions

Methods to induce fragmentation include:

1. CID or CAD

2. SID

3. Photodissociation

4. BIRD

5. ECD

Product ion nomenclature

Site of backbonecleavage

If charge is retained on amino-terminal fragment

If charge is retained on carboxy -terminal fragment

αC-C bond a x

C-N amide bond b y

N-αC bond c z

GC-MS - Gas Chromatography MS◦ separates volatile compounds in gas column and

identifies by mass

LC-MS - Liquid Chromatography MS◦ separates delicate compounds in HPLC column and

identifies by mass

MS-MS - Tandem Mass Spectrometry◦ separates compound fragments by magnetic field

and identifies by mass

Sensitivity of the procedure is determined bysensitivity of the protein purification strategyrather than the sensitivity of the MSinstrument.

Protein purification starts with a whole celllysate and ends with a gel separated proteinband /spot.

MS analysis is usually carried out on peptidesobtained after enzymatic digestion of thesegel separated proteins

Intact proteins are analyzed in special cases.

Best to minimize the number of separations

Concentration of protein : 5-50 ng

Minimize keratin contamination

Detergents and salts are incompatible.Dialysis is required.

Purity of sample.

Trypsin XXX[KR]--[P]XXX

Chymotrypsin XX[FYW]--[P]XXX

Lys C XXXXXK-- XXXXX

Asp N endo XXXXXD-- XXXXX

CNBr XXXXXM--XXXXX

K-Lysine, R-Arginine, F-Phenylalanine, Y-Tyrosine, W-Tryptophan,D-Aspartic Acid, M-Methionine, P-Proline

Robust, stable enzyme Works over a range of pH values & Temp. Quite specific and consistent in cleavage Cuts frequently to produce “ideal” MW peptides Inexpensive, easily available/purified

The peptide fragment masses provide afingerprint of the protein of interest.

Masses of measured proteolytic peptide iscompared to masses of predicted proteolyticpeptide from sequence databases.

A theoretical mass spectrum is constructedfor each protein entry from the database.

Scoring: based on mass, pI, digestionconditions, post-translational modifications.

Ranked output

Post-search analysis: Protein identification

Fig. Flow chart for the principle of Protein Mass Finger printing

First used by Gibson and Biemann in 1984.

FRAGFIT was the first computer programdeveloped for this purpose by Henzel et al in1993.

More sensitive comparisons : redundancy of64 codons is reduced to 20 amino acids.

Three types of sequence databases aresearched :

1. non – redundant protein databases

2. EST

3. genome databases.

Application URL

Eidgenossische Technische Hochschule (MassSearch)

http://cbrg.inf.ethz.ch

European Molecular Biology Laboratory (PeptideSearch)

http://www.mann.emblheidelberg.de

Swiss Institute of Bioinformatics (ExPASy)

http://www.expasy.ch/tools

Matrix Science (Mascot) http://www.matrixscience.com

Rockefeller University (PepFrag, ProFound)

http://prowl.rockefeller.edu

Human Genome Research Center (MOWSE)

http://www.seqnet.dl.ac.uk

University of California (MS-Tag, MS-Fit, MS-Seq)

http://prospector.ucsf.edu

Institute for Systems Biology (COMET)

http://www.systemsbiology.org

University of Washington (SEQUEST) http://thompson.mbt.washington.edu/sequest