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.

Highlights

  • Blood peptides Blood peptides may be identified by C18 liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) [1]

  • 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

  • The step wise organic extraction of peptides [24] provided for the enrichment of endogenous tryptic peptides with high signal to noise for random sampling [4] across disease and control treatments

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Summary

Introduction

Blood peptides Blood peptides may be identified by C18 liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) [1]. The endogenous peptides of human blood were first identified by MS/ MS fragmentation that demonstrated that a tryptic like endoproteinase activity cleaves peptides from proteins but an exopeptidase activity degraded the peptides creating a pseudo steady state [1–5]. Protein cleavage products from pro-opiomelanocortin, natriuretic peptides, insulin like growth factors, coagulation factor XIII, proglucagonderived peptides, human kallikrein-related peptidase SERPINA1, ENOSF1, neurofilament medium polypeptide, circulating IGFBP-4 fragments and many others have been suggested to have some diagnostic or mechanistic importance [7–18]. Random and independent sampling of the endogenous tryptic peptides from clinical plasma samples revealed individual analytes that show significant variation by standard statistical tests such as the Chi Square test and ANOVA [1, 2, 4, 21–23]. 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
Results
Discussion
Conclusion

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