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Clin Proteomics. 2018 Dec 21;15:41. doi: 10.1186/s12014-018-9215-z. eCollection 2018.

The plasma peptides of ovarian cancer.

Author information

1
1Ryerson Analytical Biochemistry Laboratory (RABL), Department of Chemistry and Biology, Ryerson University, Toronto, Canada.
2
2Institute of Cardiovascular Sciences, St Boniface Hospital Research Center, University of Manitoba, Winnipeg, Canada.
3
3Division of Cardiology, Department of Medicine, McMaster University, Hamilton, Canada.
4
4Keenan Chair in Medicine, St. Michael's Hospital, University of Toronto, Toronto, Canada.
5
5Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Canada.
6
6Program for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Canada.
7
7Clinical Evaluation Research Unit, Kingston General Hospital, Kingston, Canada.
8
8Alzheimer Center, Department of Neurology, Amsterdam Neuroscience, Amsterdam University Medical Centers, Vrije Universiteit, Amsterdam, The Netherlands.
9
9MS Center, Department of Neurology, Amsterdam Neuroscience, Amsterdam University Medical Centers, Vrije Universiteit, Amsterdam, The Netherlands.
10
10Neurochemistry Lab and Biobank, Department of Clinical Chemistry, Amsterdam Neuroscience, Amsterdam University Medical Centers, Vrije Universiteit, Amsterdam, The Netherlands.
11
11Mount Sinai Hospital Research Institute, University of Toronto, Toronto, Canada.
12
12University of Windsor, Windsor, Canada.
13
13International Biobank of Luxembourg (IBBL), Luxembourg Institute of Health (formerly CRP Sante Luxembourg), Strassen, Luxembourg.
14
14Department of Chemistry and Biology, Faculty of Science, Ryerson University, 350 Victoria St., Toronto, ON Canada.

Abstract

Background:

It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared to breast cancer and female cancer normal controls, other diseases with their matched or normal controls, plus ice cold plasma to control for pre-analytical variation.

Methods:

The endogenous tryptic peptides or tryptic phospho peptides (i.e. without exogenous digestion) were analyzed from 200 μl of EDTA plasma. The plasma peptides were extracted by a step gradient of organic/water with differential centrifugation, dried, and collected over C18 for analytical HPLC nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous peptides of ovarian cancer were compared to multiple disease and normal samples from different institutions alongside ice cold controls. Peptides were randomly and independently sampled by LC-ESI-MS/MS. Precursor ions from peptides > E4 counts were identified by the SEQUEST and X!TANDEM algorithms, filtered in SQL Server, before testing of frequency counts by Chi Square (χ2), for analysis with the STRING algorithm, and comparison of precursor intensity by ANOVA in the R statistical system with the Tukey-Kramer Honestly Significant Difference (HSD) test.

Results:

Peptides and/or phosphopeptides of common plasma proteins such as HPR, HP, HPX, and SERPINA1 showed increased observation frequency and/or precursor intensity in ovarian cancer. Many cellular proteins showed large changes in frequency by Chi Square (χ2 > 60, p < 0.0001) in the ovarian cancer samples such as ZNF91, ZNF254, F13A1, LOC102723511, ZNF253, QSER1, P4HA1, GPC6, LMNB2, PYGB, NBR1, CCNI2, LOC101930455, TRPM5, IGSF1, ITGB1, CHD6, SIRT1, NEFM, SKOR2, SUPT20HL1, PLCE1, CCDC148, CPSF3, MORN3, NMI, XTP11, LOC101927572, SMC5, SEMA6B, LOXL3, SEZ6L2, and DHCR24. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. Analysis of the frequently observed proteins by ANOVA confirmed increases in mean precursor intensity in ZFN91, TRPM5, SIRT1, CHD6, RIMS1, LOC101930455 (XP_005275896), CCDC37 and GIMAP4 between ovarian cancer versus normal female and other diseases or controls by the Tukey-Kramer HSD test.

Conclusion:

Here we show that separation of endogenous peptides with a step gradient of organic/water and differential centrifugation followed by random and independent sampling by LC-ESI-MS/MS with analysis of peptide frequency and intensity by SQL Server and R revealed significant difference in the ex vivo cleavage of peptides between ovarian cancer and other clinical treatments. There was striking agreement between the proteins discovered from cancer plasma versus previous biomarkers discovered in tumors by genetic or biochemical methods. The results indicate that variation in plasma proteins from ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research.

KEYWORDS:

Chi Square test and ANOVA; Discovery of variation; Electrospray ionization tandem mass spectrometry; Human EDTA plasma; LC–ESI–MS/MS; Linear quadrupole ion trap; Nano chromatography; Organic extraction; Ovarian cancer; Random and independent sampling; SQL SERVER & R

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