TY - JOUR
T1 - The plasma peptides of ovarian cancer
AU - Dufresne, Jaimie
AU - Bowden, Pete
AU - Thavarajah, Thanusi
AU - Florentinus-Mefailoski, Angelique
AU - Chen, Zhuo Zhen
AU - Tucholska, Monika
AU - Norzin, Tenzin
AU - Ho, Margaret Truc
AU - Phan, Morla
AU - Mohamed, Nargiz
AU - Ravandi, Amir
AU - Stanton, Eric
AU - Slutsky, Arthur S.
AU - Dos Santos, Claudia C.
AU - Romaschin, Alexander
AU - Marshall, John C.
AU - Addison, Christina
AU - Malone, Shawn
AU - Heyland, Daren
AU - Scheltens, Philip
AU - Killestein, Joep
AU - Teunissen, Charlotte E.
AU - Diamandis, Eleftherios P.
AU - Michael Siu, K. W.
AU - Marshall, John C.
N1 - Funding Information:
Funding to develop the SQL SERVER‑R computation platform, and to sample the breast and ovarian cancer samples, provided by the Ontario Institute of Cancer Research through the Ontario Cancer Biomarker Network to KWS, EPD, and JGM. The funding to create the reference control samples and sample the AD and MS plasma and controls was from Fonds National de la Recherche, through Luxembourg Institute of Health LIH (formerly CRP Sante) and the Integrated Biobank of Luxembourg (IBBL) to JGM. The heart attack results were collected using funding from the Heart and Stroke Foundation of Ontario and Canada to JGM. Funding for wet lab and LC–ESI–MS/MS instruments and for sampling Sepsis was from the Natural Science and Engineering Research Council of Canada (NSERC) for the CRD Grant with YYZ Pharmatech to JGM.
Funding Information:
We thank Dr. R.A. Phillips for his long running support for this program of research, his aid in obtaining human EDTA plasma from the Ontario Tumor Bank, which is funded by the Ontario Institute for Cancer Research, and his help and opinions in the preparation of the manuscript.
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/12/21
Y1 - 2018/12/21
N2 - 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 (χ 2 ), 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 (χ 2 > 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.
AB - 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 (χ 2 ), 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 (χ 2 > 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.
KW - Chi Square test and ANOVA
KW - Discovery of variation
KW - Electrospray ionization tandem mass spectrometry
KW - Human EDTA plasma
KW - LC-ESI-MS/MS
KW - Linear quadrupole ion trap
KW - Nano chromatography
KW - Organic extraction
KW - Ovarian cancer
KW - Random and independent sampling
KW - SQL SERVER & R
UR - http://www.scopus.com/inward/record.url?scp=85058930081&partnerID=8YFLogxK
U2 - 10.1186/s12014-018-9215-z
DO - 10.1186/s12014-018-9215-z
M3 - Article
AN - SCOPUS:85058930081
SN - 1542-6416
VL - 15
JO - Clinical Proteomics
JF - Clinical Proteomics
IS - 1
M1 - 41
ER -