C500 Final Report Comparison of Healthy Individuals and ...

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C500 Final Report

Comparison of Healthy Individuals and Breast Cancer

Patients Blood Sera by Liquid Chromatography-Ion-trap

Mass Spectrometry

Yuening Zhang

Department of Chemistry

Indiana Univeristy

Apr. 2006

Advisor: Dr. Milos V. Novotny

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Abstract

Breast cancer remains the leading cancer diagnosis in women in the United States. Most

breast cancers are diagnosed in advanced stages when curative treatment is impossible. Human

blood serum protein biomarkers have the potential to improve early detection of breast cancer.

This study compared the identification of three healthy and three stage II breast cancer-affected

human blood serums on LCQ and XCT Plus ion-trap LC-MS systems, respectively. It

demonstrated that the high sensitivity of XCT relative to LCQ in terms of the number of proteins

and peptides identified for the same sample. This study also compared the intensities of fourteen

proteins identified in the human blood sera of nine health individuals and six stage IV breast

cancer patients on XCT. All the fourteen proteins showed a decrease in intensities with cancer

progression. Among those, A2MG-human, APOB-human, and APOA1-human proteins showed

largest intensity change as a result of breast cancer progression.

1 Introduction

Breast cancer is a leading cause of death among women and a major problem of public

health, as suggested by the number of women who are diagnosed and die annually of this disease

[1]. Age is the most important risk factor, and the incidence of breast cancer is increasing by 0.5%

annually in the United States. Other risk factors have been reported including parity, age at the

first pregnancy, breastfeeding, age at menarche and age at menopause, estrogen treatment after

menopause, environment, stress, and nutrition. Familial history of breast cancer is another major

risk factor, emphasizing the role of genetics in this pathology [1].

The term “breast cancer” encompasses numerous types of malignant tumors which are

classified with respect to their origin and to their histological features [1]. Breast sarcomas

(malignant tumors arising from connective tissue) are rarely observed. The large majority of

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malignant breast tumors are carcinomas (malignant epithelial tumors) which are divided into two

classes: in situ carcinomas, characterized by tumor cells localized either in the ductal (ductal

carcinomas in situ, DCIS) or in the lobular (lobular carcinomas in situ, LCIS) units of the breast

cancer, without invasion through the basement membrane into the surrounding stroma; and

invasive carcinomas where the basement membrane is partially or totally destroyed. The group of

invasive carcinomas includes more than ten different types. The invasive ductal carcinomas (65-

80% of all breast cancers) and the invasive lobular carcinomas (5-15% of the cases) constitute the

two main types, while other forms such as mucinous, medullar, tubular, and apocrine carcinomas

are less frequent (0.1-4%) [1].

Molecular events leading to breast epithelial cell carcinogenesis [2] involve modification in

the structure and expression of both oncogenic and suppressive genes, leading to an unbalanced

growth characterized by high rates of proliferation and eventually migration, as well as a tendency

to better survive environmental stress which would otherwise lead to apoptosis (programmed

cellular death). However, mammary tumors develop slowly; it has been estimated that the

attainment of 1 cm in tumor size from one originating cell requires 6-8 years [1]. However, there

are currently only few markers for either early diagnosis, treatment response or for survival of

breast cancer [3]. In clinical practice, after the discovery of a tumor, its characterization as a

breast cancer is made by histology. Parameters such as tumor size and inflammation,

histoprognostic grading and node involvement are used to evaluate prognosis. It is important to

note that these parameters are only probabilistic. [1] There are currently very few molecular

markers used clinically. Thus, to improve breast cancer prognosis, it has to be diagnosed in its

early stages [4]. This can be achieved by identifying high-risk populations, enrolling them in

screening and surveillance programs, and using highly-sensitive, specific, and cost-effective

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disease markers, with the ultimate goal of either treating cancer curatively or preventing its

formation [5]. Thus the identification of markers of breast cancer development is of great

importance and would form the basis for efficient screening programs. The requirements for these

makers are the following [6]:

1. High sensitivity and specificity for the detection of cancer at an early stage or detection

of precursor lesions;

2. Easy and noninvasive access to the site of biomarker assessment, such as plama or

serum and other body fluids;

3. Cheap, rapid, reproducible, and cost-effective determination of the biomarker or

biomarker pattern;

4. The capability to tail the treatment strategy.

If these requirements were met, the diagnosis of human cancers would be greatly

facilitated, and cancer prognosis would improve. The possibility of performing large scale

proteomic analysis of human protein profiles has finally become a reality due three major events in

the biological sciences: (i) the progressive sequencing of the human genome; (ii) the development

of sensitive mass spectrometry (MS) based strategies for protein characterization; and (iii) the

concomitant development of bioinformatics, allowing the compilation of searchable genomic and

proteomic databases accessible via the internet.

Proteomics and glycoproteomics have become recently an important area of scientific

research with far-reaching potential for the fields of biochemistry, biology and medical science.

Although the evidence for definitive structural attributes of cancer cellular surface (involved in

cell “camouflage”) is sketchy, aberrant glycosylation has increasingly been associated with cancer

[7,8]. Simultaneously, while there have been numerous efforts to identify diagnostically significant

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“biomarkers” (some of these, undoubtedly, glycoproteins) through a selective, antibody-based

approach, these efforts have thus far met with only a limited success. It is thus important to

develop inclusive strategies for cancer screening and follow-up procedures.

The advancement of proteomics and glycoproteomics has been largely enhanced by the

development of instrumental capabilities of mass spectrometry (MS) that is routinely employed as

a key methodology in the structural elucidation of glycocojugates. High-sensitivity analytical

method is desirable given the limited amount of biological samples. The usefulness of Ion-trap

mass spectrometry for the structural analysis of biomolecules is now widely appreciated [9, 10].

The aim of this study is to use Ion-trap mass spectrometry coupled with nano capillary

liquid chromatography (LC) to compare glycoproteomics changes in human blood serums as a

result of cancer progression, thus potentially improve the early detection method of breast cancer.

2 Experimental

Healthy human blood sera and human blood sera from cancer patients at different cancer

stages were tryptically digested, and the resulting mixtures were analyzed by LC-Ion-trap MS.

The ability to operate ion-trap MS will permit the determination of peptides. The change in the

relative intensities of the peptides will be measured as a function of cancer progression.

2.1 Materials

Ammonia bicarbonate was purchased from Mallinckrodt Baker Inc. (Paris, KY).

Dithiothreitol (DTT) and iodoacetamide (IAA) were purchased from Bio-Rad Laboratories

(Hercules, CA). Trypsin (proteomics grade) was purchased from Sigma (Saint Louis, MO).

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Acetonitrile and water were purchased from EMD Chemicals (Gibbstown, NJ). Formic acid was

purchased from Sigma-Aldrich company (St. Louis, MO).

2.2 Sample preparation

The flowchart of sample preparation is shown in figure 1. A 30 µl aliquot from healthy

individuals and breast cancer patients blood sera were depleted through MARS (Multiple Affinity

Removal System) Hu-6 column (150 mm x 4.6 mm, Agilent, Palo Alto, CA). The buffer of the

depleted samples was exchanged three times with 2 ml of 50 mM ammonia bicarbonate. The

samples were then dried completely. A 180 µl aliquot of 100 mM ammonia bicarbonate buffer

was added to every vial, followed by 10 µl of 45 mM dithiothreitol (DTT) prepared in 100 mM

ammonium bicarbonate and incubated at 56°C for 45 mins. Next 10 µl of 100 mM iodoacetamide

(IAA) prepared in 100 mM ammonium bicarbonate was added to every sample and the solution

was kept in the dark at room temperature for 30 mins, followed by the addition of 5 µl of 1 ug/µl

trypsin (prepared in 1 mM HCl), and the solution was incubated at 37°C for

18 hrs.

2.3 Instrumentation

2.3.1 Analysis by LCQ System

Tryptically digested human serum proteome were separated on a pulled-tip capillary C18

column (150 mm x 0.075 mm) packed in-house at 1000 psi, using a properly sonicated Magic

C18AQ (Microm BioResourses, Inc., Auburn, CA) slurry of packing materials (5 µm, 120 ),

and a gas pressure chamber. The trapping columns (15 mm) were also packed in-house using

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Magic C18AQ packing slurry (5 µm, 200 ) and pre-fritted 100 µm ID fused silica capillary

(IntegraFrit, New Objective, Inc., Woburn, MA). Prior to use, the capillaries were washed with

90 % acetonitrile containing 0.1 % formic acid at 250 nL/min for 30 min, and then with 3 %

acetonitrile containing 0.1 % formic acid using an UltiMate nano-LC system (Dionex, Sunnydale,

CA). Solvent A was 0.1% formic acid and 3% acetonitrile, prepared using HPLC-grade water,

while solvent B was 0.1% formic acid prepared with HPLC-grade acetonitrile. Samples

(corresponding to 50 ng of proteins) were first loaded on the trapping column in 5% acetonitrile

and 0.1% formic acid. The sample was washed for 10 min using 3% B. Next, the sample was

separated on the capillary column using the following gradient: t= 0 min, 3% B; t= 10 min, 3% B;

t=45 min, 55%; t=50 min, 80% B; t=60 min, 80% B.

The nanoLC pulled-tip column was directly coupled to LCQ Deca XP ion-trap mass

spectrometer (ThermoElectron, San Jose, CA), which was operated in the positive-ion mode.

The instrument recorded full mass spectra (m/z 250-2000), MS2 data-dependent spectra, MS3

data-dependent spectra of those ions generated in the MS2 experiment, in the mass range

characteristic of the peptide backbone with a single N-acetylhexosamine moiety, and mass spectra

of activated ion source CID at different electrical potentials. This difference electric potential

originates from the potential gradient between the skimmer, which is at ground potential, and the

first multipole which can be set to different electric potentials. The Q activation values for all MSn

experiments were set to 0.250, while the activation times were set to 75 msec. Tandem MS

spectra were searched against protein sequences in the Swiss-Prot database using a licensed copy

of MASCOT [11] for peptide identifications.

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2.3.2 Analysis by XCT Plus System

The system comprised of XCT Plus ion trap mass spectrometer equipped with 1100 series

nano-LC pumps (Agilent, Palo Alto, CA). Tryptically digested samples were separated on a

Zorbax 300SB C18 column (3.5 µm, 150 mm x 75 µm) (Agilent, Palo Alto, CA). The trapping

column (5 mm x 300 µm) was a Pepmap 300 C18 Column (Dionex, Sunnydale, CA). Solvent A

was 0.1% formic acid and 3% acetonitrile in water, while solvent B was 0.1% formic acid in

acetonitrile. An 8 µl aliquot of the sample was first loaded on the trapping column in 3% solvent

B. The sample was washed for 10 min using 3% B. Next, the sample was separated on the

capillary column using the following gradient: t= 0 min, 5% B; t= 120 min, 45% B with the

flowrate 0.25 µl/min.

The nanoLC column was directly coupled to MSD XCT PLUS ion-trap mass

spectrometer (Agilent, Palo Alto, CA), which was operated at the positive electrospray voltage of

1600 volts. Tandem MS spectra were searched against protein sequences in the Swiss-Prot

database as described above [11].

3 Results and Discussion

3.1 Comparison of the Performance of LCQ and XCT Plus Systems

Human blood sera from three healthy individuals and three stage II breast cancer patients

were used to evaluated the performance of LCQ and XCT Plus systems.

The top five proteins with the largest number of peptides identified by LCQ and XCT

systems in healthy individuals and stage II breast cancer patients blood sera are listed in tables 1

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and 2, respectively. These five proteins were A2MG-human (alpha-2-macroglobulin precursor),

CO3-human (complement C3 precursor Containing C3a anaphylatoxin), APOA1-human

(apolipoprotein A-I precursor), CERU-human (ceruloplasmin precursor), and HEMO-human

(hemopexin precursor). The corresponding peptides for these 5 proteins are listed in tables 1and 2

according to common proteins identified by both LCQ and XCT with ion scores, unique proteins

identified by LCQ with ion scores, and unique proteins identified by XCT with ion scores. It was

demonstrated that XCT system is more sensitive and can identified more peptides for the same

protein than LCQ system. For example, in the case of the first healthy human blood serum, LCQ

identified 21 peptides for A2MG and 18 peptides for CO3, while XCT identified 41 peptides for

A2MG and 47 peptides for CO3. For the first stage II breast cancer patient blood serum, LCQ

identified 22 peptides for A2MG and 21 peptides for CO3, while XCT identified 40 peptides for

A2MG and 44 peptides for CO3.

Tables 3 and 4 list the number of amino acids and sequence coverage for selected proteins

on LCQ and XCT for healthy and stage II breast cancer patients blood sera, respectively. It could

be concluded that the sequence coverage of a particular protein attained by XCT is higher than

that attained by LCQ for the same protein originating from the same sample. For example, the

sequence coverages for the first healthy human blood serum sample are 21% for A2MG, and 16%

for CO3 on LCQ, while 40% for A2MG, and 42% for CO3 on XCT (Table 3). For the first stage

II breast cancer patient blood serum sample, the sequence coverages are 23% for A2MG, and

19% for CO3 on LCQ, while 39% for A2MG, and 41% for CO3 on XCT (Table 4). Accordingly,

XCT Plus furnishes higher sequence coverage, facilitated by the higher number of peptides

identified.

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Figure 2 compares the total proteins identified by LCQ and XCT. LCQ identified 61

proteins in total whereas XCT identified 104 proteins in total. Among these proteins, the number

of common proteins identified by both systems was 49. But the number of unique proteins

identified by LCQ was only 12, whereas the number of unique proteins identified by XCT was 55.

Figure 3 compares the total peptides identified by LCQ and XCT. LCQ identified 320

peptides in total, whereas XCT identified 661 peptides in total. Among these peptides, the number

of common peptides identified by both systems was 252. But the number of peptides which was

unique for LCQ was only 68, whereas 409 peptides were unique for XCT.

Therefore, it could be concluded that the sensitivity of the XCT system is higher than that

of the LCQ system as suggested by the higher number of identified proteins and peptides for the

same samples.

3.2 Protein Intensity Changes in Healthy Individuals and Stage IV breast cancer Patients

Blood Sera

Human blood sera from nine healthy individuals and six stage IV breast cancer patients

were used in this study utilizing the XCT system.

Figures 4 and 5 show the base peak intensity chromatograms of the tryptic digests of

blood sera proteome derived from healthy individuals and state IV breast cancer patients,

respectively, while figure 6 shows the intensity changes of fourteen selected proteins. These

proteins had largest number of peptides identified by XCT. These fourteen proteins were CO3-

human, A2MG-human, APOB-human (apolipoprotein B-100 precursor), CO4-human

(complement C4 precursor), CERU-human (ceruloplasmin precursor), APOA1-human

(apolipoprotein A-I precursor), CFAH-human (complement factor H precursor), HEMO-human

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(hemopexin precursor), CFAB-human (complement factor B precursor), ITIH2-human (inter-

alpha-trypsin inhibitor heavy chain H2 precursor), VTDB-human (vitamin D-binding protein

precursor), THRB-human (prothrombin precursor), A1BG-human (alpha-1B-glycoprotein

precursor)and KNG-human (kininogen precursor). For the fourteen selected proteins, all of them

showed decreased intensity with progression of breast cancer. Among them, A2MG-human,

APOB-human, and APOA1-human showed most obvious changes in intensities with cancer

progression. Moreover, these proteins have been previously implicated with cancer and breast

cancer [12-17].

There is some related research which showed the connection of A2MG-human [12, 13],

APOA1-human [14, 15] and APOB-human [16, 17] with cancer and breast cancer.

Kanoh and Ohtani et. al [12] reported that the levels of A2MG can predict bone

metastases in prostate cancer. They used laser-nephelometry to measure the concentration of

A2MG from the sera of 62 prostate patients. It was determined that the serum A2MG levels in

prostate cancer with bone metastases showed a significantly lower level compared with the group

without bone metastases.

Kondo and Sakaguchi et. al [13] reported the decrease of intracellular A2MG fragment

after immortalization of human cells, which is an early step in cancer development. They used

two-dimensional gel electrophoresis to compare the cellular proteins of normal and immortalized

human fibroblasts. The results demonstrated that one spot with a molecular mass of 20 kDa and

an isoelectric point of 6.0, became smaller after immortalization of human cells. Investigation of

the N-terminal amino acids revealed that the spot was a fragment of A2MG.

Maciel and Junqueira et. al [14] investigated whether serum low molecular weight

proteins can discriminate lung cancer patients from healthy donors. In their research, pooled sera

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from 20 lung cancer patients matched in sex, histology type and stage and from 20 healthy donors

were submitted to 2-D eletrophoresis coupled to MALDI-TOF peptide mass fingerprinting. The

result showed five up regulated proteins and one down regulated (fragment of apolipoprotein A-I)

in patients.

Goufman and Moshkovskii et. al [15] reported the two-dimensional electrophoretic

separation combined with MALDI-TOF mass spectrometry proteome study of serum

thermostable fraction from patients with ovarian, uterus, and breast cancers. It was found out that

apolipoprotein A-I forms have decreased spot volumes, while haptoglobin alpha 1, in contrast, is

elevated in several tumors.

Ono and Tanaka et. al [16] analyzed gene expression profiles of nine ovarian tumors using

a DNA microarray consisting of 9121 genes in order to identify genes involved in the

development and progression of ovarian cancer. Comparison of expression patterns between

carcinomas and the corresponding normal ovarian tissues enabled them to identify 55 genes that

were commonly up-regulated and 48 genes that were down-regulated (including APOB) in the

cancer specimens.

Tsai and Hancock et al [17] utilized APOB in their patent for the detection, diagnosis and

monitoring of breast cancer by comparing the protein profiles of the ductal lavage. Ductal lavage

provides a minimally invasive procedure to study the local microenvironment associated with

development and progression of breast tumors. Thus, the invention provides a sensitive method

for early detection, diagnosis and monitoring of breast cancer.

Despite these reported findings, more studies on more samples need to be performed to

confirm the connection between serum proteins and human cancers.

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4. Conclusions and Future Direction

This study compared the identification of human blood sera from three healthy individuals

and three stage II breast cancer patients on LCQ and XCT Plus systems. It showed that XCT can

identify more proteins and peptides for the same sample and is more sensitive than LCQ.

This study also compared the intensities of fourteen proteins in human blood sera from

nine healthy individuals and six stage IV breast cancer patients on XCT. All the fourteen proteins

showed decreased intensity with cancer progression. Among them, A2MG-human, APOB-human,

and APOA1-human showed largest intensity changes with breast cancer progression.

Most breast cancers are diagnosed in advanced stages when curative treatment is

impossible and prognosis is poor. Biomarkers based on the detection of certain serum proteins

that have been implicated in tumor biology have the potential to improve the early diagnosis of

human breast cancers. But mostly, the available studies were performed only with limited samples,

and the sensitivity and specificity were limited in the studies. As soon as these serum protein

profiles or biomarker patterns have been confirmed in large studies involving various centers

throughout the world, it is conceivable that serum protein profiling may allow the easy, cheap,

non invasive, reproducible, and rapid identification of breast cancer patients at high sensitivity and

specificity.

Acknowledgements

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I gratefully acknowledge Prof. Milos V. Novotny, Dr. Yehia Mechref, Milan Madera, Iveta

Klouckova, Dr. Zuzana Kyselova, Pilsoo Kang and all the other group members for their great

support and help.

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5 References

1. H. Hondermarck, A. Vercoutter-Edouart, F. Revillion, Proteomics, 2001, 1, 1216-1232.

2. D. Haber, N. Engl. J. Med., 2000, 343, 1566-1568.

3. K. Cheung, C. Graves, J. Robertson, Cancer Treat. Rev., 2000, 26, 91-102.

4. L. Bonelli, Tech. Coloproctol., 2004, 8, s267-272.

5. E. Crawford, Lancet, 2005, 365, 1447-1449.

6. M. Ebert, M. Korc, P. Malfertheiner, C. Rocken, J. Proteome Res., 2006, 5, 19-25.

7. C. Bohm, M. Mulder, R. Zennadi, M. Notter, A. Schmitt-Graff, J. Immunol. 1999, 46, 27-34.

8. E. Miyoshi, A. Nishikawa, Y. Ihara, J. Gu, Cancer Res. 1993, 53, 3899-3902.

9. A. Ivanov, L. Zang, B. Karger, Anal. Chem., 2003, 75, 5306-5316.

10. K. Zhu, J. Kim, C. Yoo, F. Miller, D. Lubman, Anal. Chem., 2003, 75, 6209-6217.

11. D. Perkins, D. Pappin, D. Creasy, J. Cottrell, Electrophoresis, 1999, 20, 3551-3567.

12. Y. Kanoh, N. Ohtani, T. Mashiko, S. Ohtani, Anticancer Res., 2001, 21(1B), 551-556.

13. T. Kondo, M. Sakaguchi, .H. Yamada, M. Namba, Electrophoresis, 1998, 19, 1836-1840.

14. C. M. Maciel, M. Junqueira, M. E. M. Paschoal, M. T. Kawamura, J. Exp. Therapeutics and

Oncology, 2004, 5, 31-38.

15. E. Goufman, S. Moshikovskii, O. Tikhonova, P. Lokhov, Biochemistry, 2006, 71, 354-360..

16. K. Ono, T. Tanaka, T. Tsunoda, O. Kitahara, Cancer Res., 2000, 60, 5007-5011.

17. J. Tsai, W. Hancock, K. Barry, PCT Int. Appl., 2005, 46pp

30 µl humanBlood Serum

Depleted HumanBlood Protein

Depleted

Buffer Exchange with50 mM NH4HCO3 (3 times),

dry

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Figure 4 Base-peak intensity chromatograms of nine healthy humanblood sera

20 30 40 50 60 70 80 90 1000.00

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Figure 5 Base-peak intensity chromatograms of six stage IV breastcancer patients blood sera

20 30 40 50 60 70 80 90 100 1100.00

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0.50

0.75

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Fig. 6 Average Protein Intensity Change with Cancer Progression

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C03 A2MG APOB CO4 CERU APOA1 CFAH HEMO CFAB ITIH2 VTDB THRB A1BG KNG

Protein

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Stage IV

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Table 1. Proteins and peptides identified by LCQ and XCT in healthy human blood seraNormal02LS09Protein Common Peptides LCQ Ion

ScoreXCT IonScore

LCQ Unique Peptides IonScore

XCT Unique Peptides IonScore

A2MG ATVLNYLPK 45 44 GHFSISIPVK 41 YGAATFTR 49 SSGSLLNNAIK 48 36 YDVENCLANK 55 TGTHGLLVK 38 SASNMAIVDVK 76 49 IAQWQSFQLEGGLK 57 FQVDNNNR 41 LPPNVVEESAR 78 51 ETTFNSLLCPSGGEVSEELSLK 64 FEVQVTVPK 48 AIGYLNTGYQR 45 66 QTVSWAVTPK 42 VGFYESDVMGR 64 66 LSFYYLIMAK 40 VTAAPQSVCALR 59 68 PVPGHVTVSICR 38 NEDSLVFVQTDK 71 75 NALFCLESAWK 54 TEHPFTVEEFVLPK 46 56 HYDGSYSTFGER 67 DTVIKPLLVEPEGLEK 70 53 DMYSFLEDMGLK 71 LLIYAVLPTGDVIGDSAK 55 43 NQGNTWLTAFVLK 92 QFSFPLSSEPFQGSYK 55 37 LVHVEEPHTETVR 42 AFQPFFVELTMPYSVIR 45 64 VTGEGCVYLQTSLK 35 VDLSFSPSQSLPASHAHLR 46 44 IAQWQSFQLEGGLK 70 VSNQTLSLFFTVLQDVPVR 70 36 TEVSSNHVLIYLDK 88 DLTGFPGPLNDQDDEDCINR 67 75 SSSNEEVMFLTVQVK 51 SLFTDLEAENDVLHCVAFAVPK 58 91 VSVQLEASPAFLAVPVEK 56

FSGQLNSHGCFYQQVK 30 LLLQQVSLPELPGEYSMK 38 PQYMVLVPSLLHTETTEK 62 YSDASDCHGEDSQAFCEK 66 KYSDASDCHGEDSQAFCEK 67 VVSMDENFHPLNELIPLVYIQDPK 43 GGVEDEVTLSAYITIALLEIPLTVTHPVVR 85

C03 FISLGEACK 35 39 LPYSVVR 30 LMNIFLK 32 YYTYLIMNK 36 62 VHQYFNVELIQPGAVK 65 ISLPESLK 32 ACEPGVDYVYK 46 58 NEQVEIR 34

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Table 1(continued)

Protein Common Peptides LCQ IonScore

XCT IonScore



TIYTPGSTVLYR 44 48 GYTQQLAFR 63 SSLSVPYVIVPLK 56 44 VLLDGVQNLR 59 IPIEDGSGEVVLSR 89 81 FYYIYNEK 33 TVMVNIENPEGIPVK 38 68 QPSSAFAAFVK 40 VFLDCCNYITELR 77 84 DFDFVPPVVR 38 VELLHNPAFCSLATTK 49 66 KVLLDGVQNLR 53 SNLDEDIIAEENIVSR 80 78 SGSDEVQVGQQ 65 SEETKENEGFTVTAEGK 55 67 KQELSEAEQATR 58 ILLQGTPVAQMTEDAVDAER 83 87 VSHSEDDCLAFK 46 AYYENSPQQVFSTEFEVK 47 59 AAVYHHFISDGVR 59 VPVAVQGEDTVQSLTQGDGVAK 68 73 GQGTLSVVTMYHAK 63 VQLSNDFDEYIMAIEQTIK 70 92 LVAYYTLIGASGQR 55 DTWVEHWPEEDECQDEENQK 55 81 RIPIEDGSGEVVLSR 61

AGDFLEANYMNLQR 59 FVTVQATFGTQVVEK 86 VYAYYNLEESCTR 78 DICEEQVNSLPGSITK 64 TELRPGETLNVNFLLR 75 EYVLPSFEVIVEPTEK 63 LSINTHPSQKPLSITVR 46 SGQSEDRQPVPGQQMTLK 38 VRVELLHNPAFCSLATTK 48 QLYNVEATSYALLALLQLK 86 YFKPGMPFDLMVFVTNPDGSPAYR 30 EGVQKEDIPPADLSDQVPDTESETR 39 QVREPGQDLVVLPLSITTDFIPSFR 36 SEFPESWLWNVEDLKEPPKNGISTK 40

APOA1 QGLLPVLESFK 50 44 AELQEGAR 33 AHVDALR 40 DLATVYVDVLK 60 77 AKPALEDLR 51 LAEYHAK 32 WQEEMELYR 44 52 ATEHLSTLSEK 48 LHELQEK 36

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Table 1(continued)


XCT IonScore



THLAPYSDELR 53 43 LEALKENGGAR 36 DYVSQFEGSALGK 77 89 VQPYLDDFQK 46 VKDLATVYVDVLK 67 83 VSFLSALEEYTK 94 LREQLGPVTQEFWDNLEK 76 65 KWQEEMELYR 47

VEPLRAELQEGAR 34 LLDNWDSVTSTFSK 102 DSGRDYVSQFEGSALGK 77 EQLGPVTQEFWDNLEK 72 EQLGPVTQEFWDNLEKETEGLR 54

52

CERU GAYPLSIEPIGVR 38 71 GVYSSDVFDIFPGTYQTLEMFPR 39 MYYSAVDPTK 31 DIASGLIGPLIICK 65 32 IYHSHIDAPK 48 EVGPTNADPVCLAK 84 54 EYTDASFTNR 44 DLYSGLIGPLIVCR 57 45 PVWLGFLGPIIK 31 KAEEEHLGILGPQLHADVGDK 62 63 QSEDSTFYLGER 44 MFTTAPDQVDKEDEDFQESNK 41 43 ALYLQYTDETFR 75 TYCSEPEKVDKDNEDFQESNR 44 58 RQSEDSTFYLGER 43 ERGPEEEHLGILGPVIWAEVGDTI

R91 46 NNEGTYYSPNYNPQSR 42

TTIEKPVWLGFLGPIIK 46 LISVDTEHSNIYLQNGPDR 43 GPEEEHLGILGPVIWAEVGDTIR 50

HEMO RLWWLDLK 44 44 NONE VDGALCMEK 41 GGYTLVSGYPK 73 55 GECQAEGVLFFQGDR 72 NFPSPVDAAFR 40 50 LYLVQGTQVYVFLTK 76 EWFWDLATGTMK 45 58 EVGTPHGIILDSVDAAFICPGSSR 35 YYCFQGNQFLR 39 59 DGWHSWPIAHQWPQGPSAVDAAFSWE

EK49

SGAQATWTELPWPHEK 48 55 SLGPNSCSANGPGLYLIHGPNLYCYSDVEK

45

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Table 1(continued)


XCT IonScore



LLQDEFPGIPSPLDAAVECHR 80 72NORMAL03L15A2MG ATVLNYLPK 43 46 VTAAPQSVCALR 56 YGAATFTR 52

SSGSLLNNAIK 39 37 TAQEGDHGSHVYTK 56 TGTHGLLVK 33 QTVSWAVTPK 60 56 ETTFNSLLCPSGGEVSEELSLK 31 FQVDNNNR 43 SASNMAIVDVK 74 56 FEVQVTVPK 47 LPPNVVEESAR 70 60 PVPGHVTVSICR 39 YDVENCLANK 67 50 DMYSFLEDMGLK 87 AIGYLNTGYQR 49 67 NQGNTWLTAFVLK 81 VGFYESDVMGR 52 58 SSSNEEVMFLTVQVK 44 NALFCLESAWK 63 42 DTVIKPLLVEPEGLEK 70 NEDSLVFVQTDK 85 79 VSVQLEASPAFLAVPVEK 57 MVSGFIPLKPTVK 47 31 FSGQLNSHGCFYQQVK 38 HYDGSYSTFGER 53 67 LLLQQVSLPELPGEYSMK 38 LVHVEEPHTETVR 31 33 VDLSFSPSQSLPASHAHLR 42 IAQWQSFQLEGGLK 55 51 MCPQLQQYEMHGPEGLR 31 TEVSSNHVLIYLDK 71 79 PQYMVLVPSLLHTETTEK 65 TEHPFTVEEFVLPK 57 66 YSDASDCHGEDSQAFCEK 53 LLIYAVLPTGDVIGDSAK 41 53 LHTEAQIQEEGTVVELTGR 72 QFSFPLSSEPFQGSYK 53 69 KYSDASDCHGEDSQAFCEK 79 AFQPFFVELTMPYSVIR 59 76 AYIFIDEAHITQALIWLSQR 41 VSNQTLSLFFTVLQDVPVR 69 51 SLFTDLEAENDVLHCVAFAVPK 93 DLTGFPGPLNDQDDEDCINR 84 58 VVSMDENFHPLNELIPLVYIQDPK 45

GGVEDEVTLSAYITIALLEIPLTVTHPVVR 85

C03 VVLVAVDK 36 35 LPYSVVR 32 GLEVTITAR 45 TGLQEVEVK 67 64 QGALELIK 57 ADIGCTPGSGK 32 FISLGEACK 55 32 LKGPLLNK 34 VLLDGVQNLR 50 GYTQQLAFR 56 64 NEQVEIR 42 FYYIYNEK 39

787

Table 1(continued)


XCT IonScore



YYTYLIMNK 48 49 DFDFVPPVVR 44 QPSSAFAAFVK 38 ACEPGVDYVYK 56 67 QPVPGQQMTLK 38 VTIKPAPETEK 35 IPIEDGSGEVVLSR 72 70 VHQYFNVELIQPGAVK 50 KVLLDGVQNLR 37 AAVYHHFISDGVR 30 59 QDSLSSQNQLGVLPLSWDIPELV

NMGQWK45 QELSEAEQATR 60

LVAYYTLIGASGQR 80 63 SGSDEVQVGQQR 71 AGDFLEANYMNLQR 60 68 APSTWLTAYVVK 52 SGIPIVTSPYQIHFTK 32 48 TIYTPGSTVLYR 70 DICEEQVNSLPGSITK 43 49 KQELSEAEQATR 69 VELLHNPAFCSLATTK 51 63 SSLSVPYVIVPLK 57 SNLDEDIIAEENIVSR 81 80 VSHSEDDCLAFK 40 TELRPGETLNVNFLLR 47 64 RAPSTWLTAYVVK 31 LSINTHPSQKPLSITVR 44 44 GQGTLSVVTMYHAK 70 QLYNVEATSYALLALLQLK 98 92 RIPIEDGSGEVVLSR 82 ILLQGTPVAQMTEDAVDAER 105 70 TVMVNIENPEGIPVK 73 AYYENSPQQVFSTEFEVK 49 53 VYAYYNLEESCTR 84 VPVAVQGEDTVQSLTQGDGVAK 78 55 VFLDCCNYITELR 83 VQLSNDFDEYIMAIEQTIK 80 65 SEETKENEGFTVTAEGK 65 DTWVEHWPEEDECQDEENQK 59 52 EYVLPSFEVIVEPTEK 59

TMQALPYSTVGNSNNYLHLSVLR 48 YFKPGMPFDLMVFVTNPDGSPAYR 39 EGVQKEDIPPADLSDQVPDTESETR 47

APOA1 AELQEGAR 30 43 LSPLGEEMR 41 AHVDALR 37 AKPALEDLR 45 34 LAEYHAK 45 ATEHLSTLSEK 44 40 LEALKENGGAR 49 QGLLPVLESFK 50 43 VQPYLDDFQK 45 DLATVYVDVLK 66 80 VSFLSALEEYTK 89 WQEEMELYR 32 48 DYVSQFEGSALGK 105 THLAPYSDELR 42 51 KWQEEMELYR 52

788

Table 1(continued)


XCT IonScore



LREQLGPVTQEFWDNLEK 68 77 VKDLATVYVDVLK 91 LLDNWDSVTSTFSK 94 EQLGPVTQEFWDNLEK 72 EQLGPVTQEFWDNLEKETEGLR 40 LREQLGPVTQEFWDNLEKETEGLR 53

CERU EYTDASFTNR 43 50 AEEEHLGILGPQLHADVGDK 46 IYHSHIDAPK 44 GAYPLSIEPIGVR 47 60 DIASGLIGPLIICK 59 QSEDSTFYLGER 47 34 ALYLQYTDETFR 37 EVGPTNADPVCLAK 67 58 DLYSGLIGPLIVCR 51 VNKDDEEFIESNK 73 31 TTIEKPVWLGFLGPIIK 46 NNEGTYYSPNYNPQSR 66 54 KAEEEHLGILGPQLHADVGDK 60 ERGPEEEHLGILGPVIWAEVGDTI

R74 51 MFTTAPDQVDKEDEDFQESNK 51

TYCSEPEKVDKDNEDFQESNR 59 GVYSSDVFDIFPGTYQTLEMFPR 37 DVDKEFYLFPTVFDENESLLLEDNIR 49

HEMO GGYTLVSGYPK 59 44 ELISER 30 LWWLDLK 31 NFPSPVDAAFR 38 56 GEFVWK 31 VDGALCMEK 47 EWFWDLATGTMK 48 61 VWVYPPEK 31 RLWWLDLK 45 YYCFQGNQFLR 44 64 QGHNSVFLIK 33 EVGTPHGIILDSVDAAFICPGSSR 32 SGAQATWTELPWPHEK 53 48 GECQAEGVLFFQGDR 79 DGWHSWPIAHQWPQGPSAVDAAFSWE

EK35

LLQDEFPGIPSPLDAAVECHR 73 63 SLGPNSCSANGPGLYLIHGPNLYCYSDVEK

62

NORMAL03LS31A2MG YGAATFTR 47 58 SSGSLLNNAIK 37 GHFSISIPVK 35

ATVLNYLPK 42 49 YDVENCLANK 68 SIYKPGQTVK 45 FEVQVTVPK 61 78 ETTFNSLLCPSGGEVSEELSLK 33 QGIPFFGQVR 36

789

Table 1(continued)


XCT IonScore



QTVSWAVTPK 69 41 LPPNVVEESAR 70 SASNMAIVDVK 74 57 LSFYYLIMAK 53 AIGYLNTGYQR 49 68 HYDGSYSTFGER 67 VGFYESDVMGR 74 72 NQGNTWLTAFVLK 70 VTAAPQSVCALR 62 67 AAQVTIQSSGTFSSK 55 NALFCLESAWK 44 47 LVHVEEPHTETVR 55 NEDSLVFVQTDK 80 72 ALLAYAFALAGNQDK 46 MVSGFIPLKPTVK 51 31 TEVSSNHVLIYLDK 85 DMYSFLEDMGLK 52 70 SSSNEEVMFLTVQVK 38 IAQWQSFQLEGGLK 69 73 QFSFPLSSEPFQGSYK 42 TEHPFTVEEFVLPK 49 60 KDTVIKPLLVEPEGLEK 47 DTVIKPLLVEPEGLEK 60 54 AFQPFFVELTMPYSVIR 55 LLIYAVLPTGDVIGDSAK 35 41 VDLSFSPSQSLPASHAHLR 83 VSVQLEASPAFLAVPVEK 68 49 YSDASDCHGEDSQAFCEK 71 LLLQQVSLPELPGEYSMK 42 38 KYSDASDCHGEDSQAFCEK 85 PQYMVLVPSLLHTETTEK 59 51 AYIFIDEAHITQALIWLSQR 39 VSNQTLSLFFTVLQDVPVR 84 33 SLFTDLEAENDVLHCVAFAVPK 105 DLTGFPGPLNDQDDEDCINR 49 47 VVSMDENFHPLNELIPLVYIQDPK 51 GGVEDEVTLSAYITIALLEIPLTVT

HPVVR49 77

CO3 DFDFVPPVVR 32 47 GVFVLNK 30 LMNIFLK 36 YYTYLIMNK 59 42 LPYSVVR 31 ADIGCTPGSGK 56 TIYTPGSTVLYR 54 46 ISLPESLK 33 GYTQQLAFR 60 SSLSVPYVIVPLK 43 53 TGLQEVEVK 69 NTLIIYLDK 32 IPIEDGSGEVVLSR 77 68 FISLGEACK 35 FYYIYNEK 32 DICEEQVNSLPGSITK 67 55 HQQTVTIPPK 53 QPSSAFAAFVK 52 SNLDEDIIAEENIVSR 69 76 VHQYFNVELIQPGAVK 63 SGSDEVQVGQQR 69 LSINTHPSQKPLSITVR 58 37 ACEPGVDYVYK 36 QLYNVEATSYALLALLQLK 108 84 KQELSEAEQATR 54

790

Table 1(continued)


XCT IonScore



ILLQGTPVAQMTEDAVDAER 118 71 AAVYHHFISDGVR 58 AYYENSPQQVFSTEFEVK 64 49 GQGTLSVVTMYHAK 63 VPVAVQGEDTVQSLTQGDGVAK 58 78 LVAYYTLIGASGQR 66 VQLSNDFDEYIMAIEQTIK 80 72 RIPIEDGSGEVVLSR 61

TVMVNIENPEGIPVK 69 AGDFLEANYMNLQR 62 VYAYYNLEESCTR 92 VFLDCCNYITELR 75 SGIPIVTSPYQIHFTK 47 VELLHNPAFCSLATTK 65 SEETKENEGFTVTAEGK 76 TELRPGETLNVNFLLR 66 EYVLPSFEVIVEPTEK 71 SGQSEDRQPVPGQQMTLK 35 VRVELLHNPAFCSLATTK 34 TMQALPYSTVGNSNNYLHLSVLR 40 DTWVEHWPEEDECQDEENQK 74 YFKPGMPFDLMVFVTNPDGSPAYR 48 EGVQKEDIPPADLSDQVPDTESETR 51 QVREPGQDLVVLPLSITTDFIPSFR 42 KHYLMWGLSSDFWGEKPNLSYIIGK 38

APOA1 AELQEGAR 38 47 LSPLGEEMR 42 LHELQEK 36 QGLLPVLESFK 51 42 ATEHLSTLSEK 61 LEALKENGGAR 53 DLATVYVDVLK 60 73 VQPYLDDFQK 51 WQEEMELYR 46 39 VSFLSALEEYTK 95 THLAPYSDELR 47 43 KWQEEMELYR 45 DYVSQFEGSALGK 82 90 LLDNWDSVTSTFSK 102 VKDLATVYVDVLK 67 83 DSGRDYVSQFEGSALGK 70 LREQLGPVTQEFWDNLEK 78 70 EQLGPVTQEFWDNLEK 72

791

Table 1(continued)


XCT IonScore



EQLGPVTQEFWDNLEKETEGLR 47 LREQLGPVTQEFWDNLEKETEGLR 54

CERU GAYPLSIEPIGVR 44 71 VNKDDEEFIESNK 67 YTVNQCR 32 QSEDSTFYLGER 68 38 ERGPEEEHLGILGPVIWAEVGDTI

R76 MYYSAVDPTK 48

DIASGLIGPLIICK 54 57 IYHSHIDAPK 45 EVGPTNADPVCLAK 53 45 DIFTGLIGPMK 53 ALYLQYTDETFR 62 75 EYTDASFTNR 42 DLYSGLIGPLIVCR 58 52 PYTFHSHGITYYK 58 TTIEKPVWLGFLGPIIK 32 56 NNEGTYYSPNYNPQSR 66 TYCSEPEKVDKDNEDFQESNR 40 56 LISVDTEHSNIYLQNGPDR 37 GVYSSDVFDIFPGTYQTLEMFPR 43 33 KAEEEHLGILGPQLHADVGDK 66

MFTTAPDQVDKEDEDFQESNK 35 SVPPSASHVAPTETFTYEWTVPK 31 HYYIAAEEIIWNYAPSGIDIFTK 49 DVDKEFYLFPTVFDENESLLLEDNIR 37

HEMO GGYTLVSGYPK 60 57 GEFVWK 30 LWWLDLK 31 NFPSPVDAAFR 53 42 QGHNSVFLIK 33 VWVYPPEK 40 EWFWDLATGTMK 49 76 RLWWLDLK 44 YYCFQGNQFLR 46 64 SGAQATWTELPWPHEK 34 LLQDEFPGIPSPLDAAVECHR 41 55 EVGTPHGIILDSVDAAFICPGSSR 46

DGWHSWPIAHQWPQGPSAVDAAFSWEEK

38

792

Table 2. Proteins and peptides identified in stage II breast cancer patients blood sera

STAGE IIOOLS07


XCT IonScore



A2MG ATVLNYLPK 54 44 GHFSISIPVK 42 SDIAPVAR 31 FEVQVTVPK 55 62 SASNMAIVDVK 67 YGAATFTR 51 QTVSWAVTPK 55 39 MVSGFIPLKPTVK 43 TGTHGLLVK 40 YDVENCLANK 54 51 ALLAYAFALAGNQDK 87 SIYKPGQTVK 32 AIGYLNTGYQR 47 65 DLTGFPGPLNDQDDEDCINR 56 LPPNVVEESAR 58 VGFYESDVMGR 61 80 QQNAQGGFSSTQDTVVALHALS

K51 LSFYYLIMAK 39

PVPGHVTVSICR 43 36 ETTFNSLLCPSGGEVSEELSLK 59 VTAAPQSVCALR 37 NQGNTWLTAFVLK 76 86 YNILPEKEEFPFALGVQTLPQTCD

EPK33 NALFCLESAWK 47

IAQWQSFQLEGGLK 63 70 NEDSLVFVQTDK 67 TEHPFTVEEFVLPK 53 51 HYDGSYSTFGER 62 DTVIKPLLVEPEGLEK 66 59 DMYSFLEDMGLK 76 VSVQLEASPAFLAVPVEK 86 48 AAQVTIQSSGTFSSK 49 LLLQQVSLPELPGEYSMK 53 37 TEVSSNHVLIYLDK 84 GGVEDEVTLSAYITIALLEIPLTVTHP

VVR42 85 SSSNEEVMFLTVQVK 32

LLIYAVLPTGDVIGDSAK 52 QFSFPLSSEPFQGSYK 35 FSGQLNSHGCFYQQVK 43 AFQPFFVELTMPYSVIR 64 VDLSFSPSQSLPASHAHLR 43 PQYMVLVPSLLHTETTEK 52 YSDASDCHGEDSQAFCEK 56 VSNQTLSLFFTVLQDVPVR 33 KYSDASDCHGEDSQAFCEK 73 AYIFIDEAHITQALIWLSQR 73 SLFTDLEAENDVLHCVAFAVPK 87 VVSMDENFHPLNELIPLVYIQDPK 49

793

Table 2 (Continued)


XCT IonScore



CO3 DFDFVPPVVR 35 36 KGYTQQLAFR 97 GLEVTITAR 45 YYTYLIMNK 44 64 VTIKPAPETEK 37 TGLQEVEVK 72

SGSDEVQVGQQR 65 58 VHQYFNVELIQPGAVK 50 NTLIIYLDK 32 ACEPGVDYVYK 50 67 VVLVSLQSGYLFIQTDK 44 FYYIYNEK 34 APSTWLTAYVVK 47 77 SEFPESWLWNVEDLKEPPK 38 HQQTVTIPPK 42 KQELSEAEQATR 38 61 QPSSAFAAFVK 33 IPIEDGSGEVVLSR 75 73 TIYTPGSTVLYR 44 VFLDCCNYITELR 88 72 VSHSEDDCLAFK 53 SGIPIVTSPYQIHFTK 32 49 AAVYHHFISDGVR 50 DICEEQVNSLPGSITK 72 52 GQGTLSVVTMYHAK 69 EYVLPSFEVIVEPTEK 70 72 RAPSTWLTAYVVK 46 LSINTHPSQKPLSITVR 57 62 LVAYYTLIGASGQR 64 QLYNVEATSYALLALLQLK 103 87 RIPIEDGSGEVVLSR 54 ILLQGTPVAQMTEDAVDAER 90 75 TVMVNIENPEGIPVK 69 AYYENSPQQVFSTEFEVK 59 47 VYAYYNLEESCTR 77 VPVAVQGEDTVQSLTQGDGVAK 90 78 VELLHNPAFCSLATTK 68

SNLDEDIIAEENIVSR 75 KVFLDCCNYITELR 70 SEETKENEGFTVTAEGK 66 TELRPGETLNVNFLLR 68 VRVELLHNPAFCSLATTK 48 VQLSNDFDEYIMAIEQTIK 78 TMQALPYSTVGNSNNYLHLSVLR 43 DTWVEHWPEEDECQDEENQK 83 YFKPGMPFDLMVFVTNPDGSPAYR 39 EGVQKEDIPPADLSDQVPDTESETR 50 QVREPGQDLVVLPLSITTDFIPSFR 46 SEFPESWLWNVEDLKEPPKNGIST

K39

794

Table 2 (Continued)


XCT IonScore



APOA1 QGLLPVLESFK 53 38 LAEYHAK 32 AHVDALR 34 DLATVYVDVLK 55 77 AELQEGAR 38 VQPYLDDFQK 45 WQEEMELYR 40 38 LHELQEK 33 DYVSQFEGSALGK 93 THLAPYSDELR 30 52 LLDNWDSVTSTFSK 88 VSFLSALEEYTK 72 89 EQLGPVTQEFWDNLEK 61 VKDLATVYVDVLK 60 57 LREQLGPVTQEFWDNLEKETEGLR 42 LREQLGPVTQEFWDNLEK 70 68

CERU DIASGLIGPLIICK 68 62 GVYSSDVFDIFPGTYQTLEMFPR 57 EYTDASFTNR 52 EVGPTNADPVCLAK 67 54 GAYPLSIEPIGVR 55 ALYLQYTDETFR 53 70 TTIEKPVWLGFLGPIIK 52 DLYSGLIGPLIVCR 54 67 AEEEHLGILGPQLHADVGDK 31 NNEGTYYSPNYNPQSR 67 54 LISVDTEHSNIYLQNGPDR 37 GPEEEHLGILGPVIWAEVGDTIR 42 44 KAEEEHLGILGPQLHADVGDK 91 ERGPEEEHLGILGPVIWAEVGDTIR 82 64 MFTTAPDQVDKEDEDFQESNK 61

TYCSEPEKVDKDNEDFQESNR 60 HYYIAAEEIIWNYAPSGIDIFTK 50 DVDKEFYLFPTVFDENESLLLEDNIR 30

HEMO GGYTLVSGYPK 57 49 NONE VDGALCMEK 34 EWFWDLATGTMK 48 70 NFPSPVDAAFR 67 SGAQATWTELPWPHEK 51 46 YYCFQGNQFLR 59 LLQDEFPGIPSPLDAAVECHR 43 61 EVGTPHGIILDSVDAAFICPGSSR 37

DGWHSWPIAHQWPQGPSAVDAAFSWEEK

31

SLGPNSCSANGPGLYLIHGPNLYCYSDVEK

59

STAGE I I00LS08

795

Table 2 (Continued)


XCT IonScore



A2MG ATVLNYLPK 53 49 GHFSISIPVK 41 YGAATFTR 52 FEVQVTVPK 63 63 ETTFNSLLCPSGGEVSEELSLK 43 SIYKPGQTVK 34 QTVSWAVTPK 63 41 QGIPFFGQVR 37 SASNMAIVDVK 65 76 LPPNVVEESAR 60 YDVENCLANK 54 46 LSFYYLIMAK 30 AIGYLNTGYQR 50 74 VGFYESDVMGR 75 VTAAPQSVCALR 59 63 PVPGHVTVSICR 40 NEDSLVFVQTDK 84 78 NALFCLESAWK 57 MVSGFIPLKPTVK 64 37 HYDGSYSTFGER 71 DMYSFLEDMGLK 51 83 NQGNTWLTAFVLK 97 IAQWQSFQLEGGLK 57 86 AAQVTIQSSGTFSSK 34 TEHPFTVEEFVLPK 44 65 LVHVEEPHTETVR 34 DTVIKPLLVEPEGLEK 34 63 ALLAYAFALAGNQDK 60 LLIYAVLPTGDVIGDSAK 53 57 TEVSSNHVLIYLDK 87 QFSFPLSSEPFQGSYK 58 50 SSSNEEVMFLTVQVK 42 VSVQLEASPAFLAVPVEK 73 51 FSGQLNSHGCFYQQVK 40 LLLQQVSLPELPGEYSMK 49 42 AFQPFFVELTMPYSVIR 53 VSNQTLSLFFTVLQDVPVR 65 38 VDLSFSPSQSLPASHAHLR 52 DLTGFPGPLNDQDDEDCINR 59 65 PQYMVLVPSLLHTETTEK 73

YSDASDCHGEDSQAFCEK 61 HNVYINGITYTPVSSTNEK 42 KYSDASDCHGEDSQAFCEK 79 AYIFIDEAHITQALIWLSQR 44 SLFTDLEAENDVLHCVAFAVPK 94 VVSMDENFHPLNELIPLVYIQDPK 43 GGVEDEVTLSAYITIALLEIPLTVTHP

VVR97

CO3 YYTYLIMNK 42 64 KQELSEAEQATR 42 LPYSVVR 34 ACEPGVDYVYK 43 67 VHQYFNVELIQPGAVK 71 LMNIFLK 38 IPIEDGSGEVVLSR 86 77 EPGQDLVVLPLSITTDFIPSFR 66 GLEVTITAR 69

796

Table 2 (Continued)


XCT IonScore



AAVYHHFISDGVR 31 51 GYTQQLAFR 40 DICEEQVNSLPGSITK 69 57 VLLDGVQNLR 63 VELLHNPAFCSLATTK 46 64 FYYIYNEK 35 SNLDEDIIAEENIVSR 74 71 QPSSAFAAFVK 39 EYVLPSFEVIVEPTEK 79 76 DFDFVPPVVR 39 QLYNVEATSYALLALLQLK 102 93 ENEGFTVTAEGK 40 ILLQGTPVAQMTEDAVDAER 92 72 SGSDEVQVGQQR 80 AYYENSPQQVFSTEFEVK 56 52 APSTWLTAYVVK 73 VPVAVQGEDTVQSLTQGDGVAK 67 71 TIYTPGSTVLYR 56 VQLSNDFDEYIMAIEQTIK 58 71 SSLSVPYVIVPLK 42

VSHSEDDCLAFK 45 GQGTLSVVTMYHAK 52 LVAYYTLIGASGQR 59 RIPIEDGSGEVVLSR 59 TVMVNIENPEGIPVK 46 AGDFLEANYMNLQR 69 VYAYYNLEESCTR 98 KVFLDCCNYITELR 63 SEETKENEGFTVTAEGK 63 TELRPGETLNVNFLLR 66 LSINTHPSQKPLSITVR 61 VRVELLHNPAFCSLATTK 48 TMQALPYSTVGNSNNYLHLSVLR 30 DTWVEHWPEEDECQDEENQK 92 YFKPGMPFDLMVFVTNPDGSPAYR 52 EGVQKEDIPPADLSDQVPDTESETR 47 QVREPGQDLVVLPLSITTDFIPSFR 47

APOA1 ATEHLSTLSEK 43 39 LHELQEK 34 AELQEGAR 33 QGLLPVLESFK 52 45 LSPLGEEMR 44 LEALKENGGAR 47

797

Table 2 (Continued)


XCT IonScore



DLATVYVDVLK 59 86 VQPYLDDFQK 44 WQEEMELYR 30 52 KWQEEMELYR 54 THLAPYSDELR 43 48 VEPLRAELQEGAR 31 VSFLSALEEYTK 79 94 LLDNWDSVTSTFSK 102 DYVSQFEGSALGK 82 90 DSGRDYVSQFEGSALGK 79 VKDLATVYVDVLK 71 83 EQLGPVTQEFWDNLEK 73 LREQLGPVTQEFWDNLEK 79 74 EQLGPVTQEFWDNLEKETEGLR 54

LREQLGPVTQEFWDNLEKETEGLR 54

CERU DIASGLIGPLIICK 61 41 NONE IYHSHIDAPK 45 EVGPTNADPVCLAK 71 57 EYTDASFTNR 46 DLYSGLIGPLIVCR 74 70 PVWLGFLGPIIK 49 NNEGTYYSPNYNPQSR 47 54 GAYPLSIEPIGVR 60 TTIEKPVWLGFLGPIIK 35 58 ALYLQYTDETFR 70 ERGPEEEHLGILGPVIWAEVGDTIR 77 34 LISVDTEHSNIYLQNGPDR 34

KAEEEHLGILGPQLHADVGDK 85 MFTTAPDQVDKEDEDFQESNK 56 TYCSEPEKVDKDNEDFQESNR 65 GVYSSDVFDIFPGTYQTLEMFPR 34

HEMO GGYTLVSGYPK 62 44 DYFMPCPGR 32 LWWLDLK 31 NFPSPVDAAFR 42 40 VDGALCMEK 42 EWFWDLATGTMK 52 66 RLWWLDLK 45 SGAQATWTELPWPHEK 60 49 YYCFQGNQFLR 64 LLQDEFPGIPSPLDAAVECHR 73 80 DGWHSWPIAHQWPQGPSAVDAAF

SWEEK43

EVGTPHGIILDSVDAAFICPGSSR 63 40 SLGPNSCSANGPGLYLIHGPNLYCYSDVEK

44

STAGEII00LS10A2MG ATVLNYLPK 53 45 GHFSISIPVK 41 YGAATFTR 49

798

Table 2 (Continued)


XCT IonScore



SASNMAIVDVK 79 56 SSGSLLNNAIK 59 FEVQVTVPK 68 LPPNVVEESAR 79 64 QTVSWAVTPK 57 LSFYYLIMAK 55 YDVENCLANK 73 47 QGIPFFGQVR 39 NALFCLESAWK 49 AIGYLNTGYQR 50 66 MVSGFIPLKPTVK 44 HYDGSYSTFGER 63 VGFYESDVMGR 68 73 DMYSFLEDMGLK 59 VTAAPQSVCALR 59 65 AAQVTIQSSGTFSSK 69 NEDSLVFVQTDK 73 82 VTGEGCVYLQTSLK 31 NQGNTWLTAFVLK 89 86 ALLAYAFALAGNQDK 66 LVHVEEPHTETVR 31 38 TEVSSNHVLIYLDK 73 IAQWQSFQLEGGLK 67 53 SSSNEEVMFLTVQVK 66 TEHPFTVEEFVLPK 38 50 DTVIKPLLVEPEGLEK 61 LLIYAVLPTGDVIGDSAK 34 33 GHFSISIPVKSDIAPVAR 36 QFSFPLSSEPFQGSYK 51 56 FSGQLNSHGCFYQQVK 50 VSVQLEASPAFLAVPVEK 50 52 AFQPFFVELTMPYSVIR 56 VDLSFSPSQSLPASHAHLR 47 43 LLLQQVSLPELPGEYSMK 37 SLFTDLEAENDVLHCVAFAVPK 41 85 YSDASDCHGEDSQAFCEK 67 GGVEDEVTLSAYITIALLEIPLTVTHP

VVR34 85 LHTEAQIQEEGTVVELTGR 43

HNVYINGITYTPVSSTNEK 31 VSNQTLSLFFTVLQDVPVR 40 KYSDASDCHGEDSQAFCEK 63 AYIFIDEAHITQALIWLSQR 37 AFQPFFVELTMPYSVIRGEAFTLK 57 VVSMDENFHPLNELIPLVYIQDPK 41

CO3 QPSSAFAAFVK 36 44 LPYSVVR 33 GLEVTITAR 38 DFDFVPPVVR 34 37 ISLPESLK 31 FISLGEACK 41 ACEPGVDYVYK 52 61 FYYIYNEK 32 YYTYLIMNK 41 TIYTPGSTVLYR 52 54 KGYTQQLAFR 89 NTMILEICTR 52 IPIEDGSGEVVLSR 80 85 VFLDCCNYITELR 57 SSLSVPYVIVPLK 35 AGDFLEANYMNLQR 42 63 VHQYFNVELIQPGAVK 30 AAVYHHFISDGVR 57

799

Table 2 (Continued)


XCT IonScore



VYAYYNLEESCTR 43 73 TELRPGETLNVNFLLR 38 GQGTLSVVTMYHAK 44 SGIPIVTSPYQIHFTK 47 52 SSLSVPYVIVPLKTGLQEVEVK 40 LVAYYTLIGASGQR 78 DICEEQVNSLPGSITK 48 52 EPGQDLVVLPLSITTDFIPSFR 67 RIPIEDGSGEVVLSR 43 SNLDEDIIAEENIVSR 85 80 LDKACEPGVDYVYK 40 QLYNVEATSYALLALLQLK 102 62 VELLHNPAFCSLATTK 39 ILLQGTPVAQMTEDAVDAER 98 80 SEETKENEGFTVTAEGK 55 AYYENSPQQVFSTEFEVK 81 51 EYVLPSFEVIVEPTEK 54 VPVAVQGEDTVQSLTQGDGVAK 63 85 LSINTHPSQKPLSITVR 43 VQLSNDFDEYIMAIEQTIK 80 67 VRVELLHNPAFCSLATTK 47 DTWVEHWPEEDECQDEENQK 48 58 TMQALPYSTVGNSNNYLHLSVLR 32 EGVQKEDIPPADLSDQVPDTESETR 31 54 YFKPGMPFDLMVFVTNPDGSPAYR 36

QVREPGQDLVVLPLSITTDFIPSFR 56 KHYLMWGLSSDFWGEKPNLSYIIG

K54

QLYNVEATSYALLALLQLKDFDFVPPVVR

30

APOA1 WQEEMELYR 45 46 LSPLGEEMR 34 LEALKENGGAR 55 THLAPYSDELR 53 39 ATEHLSTLSEK 41 QGLLPVLESFK 47 VEPLRAELQEGAR 31 38 VQPYLDDFQK 36 DLATVYVDVLK 83 DLATVYVDVLKDSGR 54 51 VSFLSALEEYTK 85 DSGRDYVSQFEGSALGK 69 82 DYVSQFEGSALGK 101 LREQLGPVTQEFWDNLEKETEGLR 56 68 VKDLATVYVDVLK 78

THLAPYSDELRQR 37 LLDNWDSVTSTFSK 88 VKDLATVYVDVLKDSGR 74 EQLGPVTQEFWDNLEK 56 LREQLGPVTQEFWDNLEK 63 EQLGPVTQEFWDNLEKETEGLR 33 DLATVYVDVLKDSGRDYVSQFEGS

ALGK44

800

Table 2 (Continued)


XCT IonScore



CERU GAYPLSIEPIGVR 65 67 EYTDASFTNR 62 IYHSHIDAPK 44 QSEDSTFYLGER 30 66 NLASRPYTFHSHGITYYK 36 ALYLQYTDETFR 71 DIASGLIGPLIICK 70 57 DLYSGLIGPLIVCR 55 EVGPTNADPVCLAK 89 58 LISVDTEHSNIYLQNGPDR 35 NNEGTYYSPNYNPQSR 80 65 KAEEEHLGILGPQLHADVGDK 67 TTIEKPVWLGFLGPIIK 34 44 FNKNNEGTYYSPNYNPQSR 40 TYCSEPEKVDKDNEDFQESNR 45 60 MFTTAPDQVDKEDEDFQESNK 50 GVYSSDVFDIFPGTYQTLEMFPR 64 33 KAEEEHLGILGPQLHADVGDKVK 56

TYSDHPEKVNKDDEEFIESNK 43 SVPPSASHVAPTETFTYEWTVPK 32 HYYIAAEEIIWNYAPSGIDIFTK 54 ERGPEEEHLGILGPVIWAEVGDTIR 49

HEMO RLWWLDLK 37 45 GGYTLVSGYPK 60 LWWLDLK 31 NFPSPVDAAFR 38 51 GECQAEGVLFFQGDR 67 YYCFQGNQFLR 42 66 EVGTPHGIILDSVDAAFICPGSSR 37 WKNFPSPVDAAFR 61 41 SGAQATWTELPWPHEKVDGALCM

EK38

SGAQATWTELPWPHEK 69 54 GECQAEGVLFFQGDREWFWDLATGTMK

66

LLQDEFPGIPSPLDAAVECHR 58 37 DGWHSWPIAHQWPQGPSAVDAAFSWEEK

49

SLGPNSCSANGPGLYLIHGPNLYCYSDVEK

37

801

Table 3 Selected protein sequence coverages for healthy human blood sera on LCQ and XCT(AA for amino acid, SC for sequence coverage)

Protein Name TotalAA

LCQNormal

1

SC(%) XCTNormal

1

SC(%) LCQNormal

2

SC(%) XCTNormal

2

SC(%) LCQNormal

3

SC(%)

XCTNormal

3

SC(%)

A2MG_HUMAN 1474 305 20.69 594 40.30 325 22.05 607 41.18 357 24.22 634 43.01

CO3_HUMAN 1663 267 16.06 703 42.27 430 25.86 668 40.17 271 16.30 665 39.99

APOA1_HUMAN 267 114 42.70 256 95.88 97 36.33 247 92.51 114 42.70 237 88.76

CERU_HUMAN 1065 116 10.89 297 27.89 123 11.55 282 26.48 178 16.71 350 32.86

HEMO_HUMAN 462 90 19.48 211 45.67 127 27.49 188 40.69 82 17.75 129 27.92

PZP_HUMAN 1482 0 0.00 0 0.00 0 0.00 71 4.79 0 0.00 60 4.05

VTDB_HUMAN 474 78 16.46 127 26.79 44 9.28 116 24.47 78 16.46 102 21.52

APOB_HUMAN 4563 96 2.10 253 5.54 53 1.16 314 6.88 61 1.34 147 3.22

ITIH2_HUMAN 946 60 6.34 126 13.32 26 2.75 126 13.32 90 9.51 101 10.68

APOH_HUMAN 345 70 20.29 96 27.83 15 4.35 75 21.74 14 4.06 75 21.74

A1BG_HUMAN 495 40 8.08 112 22.63 29 5.86 156 31.52 12 2.42 118 23.84

HRG_HUMAN 525 0 0.00 42 8.00 44 8.38 51 9.71 0 0.00 56 10.67

A1AG1_HUMAN 201 26 12.94 97 48.26 55 27.36 47 23.38 17 8.46 70 34.83

CFAB_HUMAN 764 44 5.76 105 13.74 14 1.83 91 11.91 38 4.97 101 13.22

A1AG2_HUMAN 201 27 13.43 61 30.35 49 24.38 41 20.40 25 12.44 34 16.92

ANGT_HUMAN 485 53 10.93 70 14.43 0 0.00 54 11.13 29 5.98 95 19.59

CO4_HUMAN 1744 50 2.87 324 18.58 111 6.36 367 21.04 26 1.49 111 6.36

802

Table 3 (Continued)

Protein Name TotalAA

LCQNormal

1

SC(%) XCTNormal

1

SC(%) LCQNormal

2

SC(%) XCTNormal

2

SC(%) LCQNormal

3

SC(%)

XCTNormal

3

SC(%)

APOA2_HUMAN 100 56 56.00 72 72.00 95 95.00 62 62.00 54 54.00 62 62.00

THRB_HUMAN 622 55 8.84 96 15.43 0 0.00 105 16.88 19 3.05 78 12.54

ITIH1_HUMAN 911 56 6.15 133 14.60 19 2.09 125 13.72 37 4.06 106 11.64

PON1_HUMAN 354 0 0.00 16 4.52 0 0.00 38 10.73 0 0.00 26 7.34

GELS_HUMAN 782 0 0.00 35 4.48 0 0.00 39 4.99 0 0.00 35 4.48

APOA_HUMAN 4548 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00

AFAM_HUMAN 599 0 0.00 39 6.51 0 0.00 36 6.01 0 0.00 0 0.00

HEP2_HUMAN 499 0 0.00 27 5.41 0 0.00 0 0.00 0 0.00 11 2.20

PGRP2_HUMAN 576 0 0.00 36 6.25 0 0.00 38 6.60 0 0.00 0 0.00

APOD_HUMAN 189 0 0.00 25 13.23 0 0.00 11 5.82 0 0.00 0 0.00

FHR1_HUMAN 330 0 0.00 24 7.27 0 0.00 0 0.00 0 0.00 0 0.00

CO8A_HUMAN 584 0 0.00 34 5.82 0 0.00 0 0.00 0 0.00 17 2.91

CO8G_HUMAN 202 0 0.00 32 15.84 0 0.00 0 0.00 0 0.00 0 0.00

FETUA_HUMAN 367 28 7.63 45 12.26 28 7.63 31 8.45 28 7.63 35 9.54

VTNC_HUMAN 478 22 4.60 43 9.00 27 5.65 27 5.65 27 5.65 67 14.02

APOC3_HUMAN 99 27 27.27 27 27.27 16 16.16 27 27.27 27 27.27 27 27.27

TTHY_HUMAN 147 27 18.37 24 16.33 36 24.49 0 0.00 0 0.00 0 0.00

KNG_HUMAN 644 19 2.95 112 17.39 14 2.17 109 16.93 19 2.95 118 18.32

803

Table 4 Selected protein sequence coverages for stage II breast cancer patients blood sera on LCQ andXCT (AA for amino acid, SC for sequence coverage)

Protein Total AA LCQ S1 SC(%) XCT S1 SC(%) LCQ S2 SC(%) XCT S2 SC(%) LCQ S3 SC(%) XCT S3 SC(%)

A2MG_HUMAN 1474 336 22.80 580 39.35 294 19.95 664 45.05 318 21.57 646 43.83

CO3_HUMAN 1663 310 18.64 682 41.01 259 15.57 637 38.30 398 23.93 587 35.30

APOA1_HUMAN 267 107 40.07 151 56.55 125 46.82 216 80.90 119 44.57 226 84.64

CERU_HUMAN 1065 141 13.24 309 29.01 100 9.39 262 24.60 158 14.84 361 33.90

HEMO_HUMAN 462 60 12.99 173 37.45 104 22.51 188 40.69 91 19.70 221 47.84

PZP_HUMAN 1482 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00

VTDB_HUMAN 474 60 12.66 159 33.54 90 18.99 161 33.97 49 10.34 203 42.83

APOB_HUMAN 4563 84 1.84 266 5.83 243 5.33 682 14.95 132 2.89 359 7.87

ITIH2_HUMAN 946 45 4.76 101 10.68 68 7.19 100 10.57 15 1.59 111 11.73

APOH_HUMAN 345 0 0.00 75 21.74 14 4.06 75 21.74 15 4.35 85 24.64

A1BG_HUMAN 495 60 12.12 118 23.84 34 6.87 108 21.82 39 7.88 141 28.48

HRG_HUMAN 525 21 4.00 54 10.29 0 0.00 58 11.05 51 9.71 65 12.38

A1AG1_HUMAN 201 48 23.88 74 36.82 23 11.44 74 36.82 33 16.42 47 23.38

CFAB_HUMAN 764 42 5.50 121 15.84 18 2.36 96 12.57 79 10.34 127 16.62

A1AG2_HUMAN 201 36 17.91 68 33.83 41 20.40 53 26.37 28 13.93 53 26.37

ANGT_HUMAN 485 29 5.98 70 14.43 29 5.98 71 14.64 0 0.00 99 20.41

CO4_HUMAN 1744 41 2.35 241 13.82 115 6.59 283 16.23 41 2.35 240 13.76

APOA2_HUMAN 100 52 52.00 62 62.00 85 85.00 72 72.00 33 33.00 58 58.00

804

Table 4 (Continued)

Protein Total AA LCQ S1 SC(%) XCT S1 SC(%) LCQ S2 SC(%) XCT S2 SC(%) LCQ S3 SC(%) XCT S3 SC(%)

THRB_HUMAN 622 19 3.05 120 19.29 31 4.98 153 24.60 19 3.05 134 21.54

ITIH1_HUMAN 911 37 4.06 136 14.93 19 2.09 93 10.21 19 2.09 108 11.86

PON1_HUMAN 354 0 0.00 31 8.76 0 0.00 38 10.73 0 0.00 30 8.47

GELS_HUMAN 782 0 0.00 75 9.59 0 0.00 35 4.48 0 0.00 30 3.84

APOA_HUMAN 4548 23 0.51 20 0.44 0 0.00 0 0.00 0 0.00 0 0.00

AFAM_HUMAN 599 0 0.00 0 0.00 0 0.00 48 8.01 0 0.00 31 5.18

HEP2_HUMAN 499 0 0.00 38 7.62 0 0.00 27 5.41 0 0.00 0 0.00

PGRP2_HUMAN 576 0 0.00 0 0.00 0 0.00 72 12.50 0 0.00 0 0.00

APOD_HUMAN 189 0 0.00 21 11.11 0 0.00 25 13.23 0 0.00 0 0.00

FHR1_HUMAN 330 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00

CO8A_HUMAN 584 0 0.00 0 0.00 0 0.00 17 2.91 0 0.00 0 0.00

CO8G_HUMAN 202 0 0.00 15 7.43 0 0.00 0 0.00 0 0.00 0 0.00

FETUA_HUMAN 367 41 11.17 45 12.26 41 11.17 76 20.71 28 7.63 55 14.99

VTNC_HUMAN 478 27 5.65 53 11.09 12 2.51 58 12.13 15 3.14 58 12.13

APOC3_HUMAN 99 16 16.16 27 27.27 16 16.16 27 27.27 16 16.16 30 30.30

TTHY_HUMAN 147 13 8.84 0 0.00 13 8.84 0 0.00 23 15.65 0 0.00

FA12_HUMAN 615 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00

SNTB1_HUMAN 537 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00

KNG_HUMAN 644 0 0.00 121 18.79 14 2.17 107 16.61 34 5.28 117 18.17

805

Table 5 Selected protein intensities in healthy human blood sera

Protein N00LS13 N01LS01 N01LS06 N02LS01 N03LS35 N03LS38 N02LS09 N03LS15 N03LS31 N AVE

C03 200375993 326739844 473275054 409338011 316031263 559593113 504261564 439897155 421979176 405721241

A2MG 479505104 542283170 1101841055 984905458 311982900 951810226 420033466 477632708 459527375 636613496

APOB 115973628 168605316 401691565 267454402 219094661 225780427 104511009 130892460 62738485 188526884

CO4 182845947 198057942 315506403 241760715 199139826 577998458 184769073 180687533 32574661 234815618

CERU 136219621 247642326 415817160 244957881 130856162 296284783 152212239 150197530 216568632 221195148

APOA1 572717683 815436039 1285962388 1.039E+09 355626263 775553611 752416591 541842871 470086089 734347052

CFAH 54130809 219677997 197381543 111283611 78593230 141979145 78236711 73266353 7779505 106925434

HEMO 106717997 229614241 362993430 310733938 221997610 286457467 142359904 173509485 114433321 216535266

CFAB 63087254 147943735 83167983 66874096 83218117 128249051 52072880 42422945 30903096 77548795

ITIH2 68083589 116773921 199876504 227806889 114239966 123801838 38842542 62115691 37170809 109856861

VTDB 124976657 226082982 317359862 284334559 126590196 173732306 72843277 98304252 106970664 170132751

THRB 53537419 167933447 128864910 146083240 132253939 99154411 40692553 57841077 33851064 95579118

A1BG 112496194 158250750 251465620 199076734 98268793 180009981 49497942 93531625 58814763 133490267

KNG 49236160 51198380 170849049 63590542 54239220 263789632 76194991 81344922 46651172 95232674

806

Table 6 Selected protein intensities in stage IV breast cancer patientsblood sera

Protein LIS 444 BC DEH DGF LAH IV AVE

C03 226289534 325395061 595218059 333383747 604529336 269674172 392414985

A2MG 242627916 438013137 631511889 409408004 476966589 486981209 447584791

APOB 21209995 72277235 0 0 0 44073365 22926765.8

CO4 137398202 116774872 372113778 186058680 308482788 111841177 205444916

CERU 123525239 60907356 178894554 181512453 146701737 136965348 138084448

APOA1 147301646 139492942 786086026 446957374 291843860 279100521 348463728

CFAH 32624706 30946017 159870708 48407892 64466170 19869996 59364248.2

HEMO 56262141 74024335 247458361 152371949 139834277 70714794 123444310

CFAB 42056796 14567708 120917976 83130290 59096981 44954014 60787294.2

ITIH2 52986904 9655801 119755236 40715582 62812472 37349828 53879303.8

VTDB 47087168 33021204 211002373 104795382 39971813 59095643 82495597.2

THRB 19843374 30456450 122054773 60194170 66219024 35779339 55757855

A1BG 56254782 49263619 144647031 71523490 96542484 60110918 79723720.7

KNG 18576854 29573001 82267520 40390088 55613412 8515269 39156024

807

Appendix A: FoxO1 (1-334) nucleotide sequence

1 atggccgagg cgcctcaggt ggtggagatc gacccggact tcgagccgct gccccggccg

61 cgctcgtgca cctggccgct gcccaggccg gagtttagcc agtccaactc ggccacctcc

121 agcccggcgc cgtcgggcag cgcggctgcc aaccccgacg ccgcggcggg cctgccctcg

181 gcctcggctg ccgctgtcag cgccgacttc atgagcaacc tgagcttgct ggaggagagc

241 gaggacttcc cgcaggcgcc cggctccgtg gcggcggcgg tggcggcggc ggccgccgcg

301 gccgccaccg gggggctgtg cggggacttc cagggcccgg aggcgggctg cctgcaccca

361 gcgccaccgc agcccccgcc gcccgggccg ctgtcgcagc acccgccggt gccccccgcc

421 gccgctgggc cgctcgcggg gcagccgcgc aagagcagct cgtcccgccg caacgcgtgg

481 ggcaacctgt cctacgccga cctcatcacc aaggccatcg agagctcggc ggagaagcgg

541 ctcacgctgt cgcagatcta cgagtggatg gtcaagagcg tgccctactt caaggataag

601 ggtgacagca acagctcggc gggctggaag aattcaattc gtcataatct gtccctacac

661 agcaagttca ttcgtgtgca gaatgaagga actggaaaaa gttcttggtg gatgctcaat

721 ccagagggtg gcaagagcgg gaaatctcct aggagaagag ctgcatccat ggacaacaac

781 agtaaatttg ctaagagccg aagccgagct gccaagaaga aagcatctct ccagtctggc

841 caggagggtg ctggggacag ccctggatca cagttttcca aatggcctgc aagccctggc

901 tctcacagca atgatgactt tgataactgg agtacatttc gccctcgaac tagctcaaat

961 gctagtacta ttagtgggag actctcaccc attatgaccg aatga

Targeted mutagenesis sites:

Threonine 24 Alanine Nucleotide: 70, 71, and 72 Codon: ACC (Thr)

Serine 256 Alanine Nucleotide: 766, 767, and 768 Codon: TCC (Ser)

Serine 319 Alanine Nucleotide: 955, 956, and 957 Codon: TCA (Ser)

808

Appendix B: Oligonucleotide Primer Design

Threonine 24 to Alanine: T24A

ccgctgccccggccgcgctcgtgcacctggccgctgcccaggccg

45 bp (46-90) ACC = T24

ccgctgccccggccgcgctcgtgcacctggccgctgcccaggccg

Alanine: GCU(T), GCC, GCA, GCG

Primer:

Sense: GCTGCCCCGGCCGCGCTCGTAntisense: GGCCTGGGCAGCGGCCAGGT

Serine 256 to Alanine: S256A

gcgggctggaagaattcaattcgtcataatctgtccctacac

42 bp (619-660) TCA = S256

gcgggctggaagaattcaattcgtcataatctgtccctacac


Primer:

Sense: GCGGGCTGGAAGAATTCAATAntisense: GTGTAGGGACAGATTATGAC

Serine 319 to Alanine: S319A

aggagaagagctgcatccatggacaacaacagtaaatttgct

42 bp (751-792) TCA = S319

aggagaagagctgcatccatggacaacaacagtaaatttgct


Primer:

Sense: AGGAGAAGAGCTGCATCCATAntisense: GCAAATTTACTGTTGTTGTC

C500 Final Report Comparison of Healthy Individuals and ...

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