Peptidomic analysis of human blood specimens: Comparison between plasma specimens and serum by differential peptide display

Abstract

The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.

Analysis of human serum by liquid chromatography–mass spectrometry: Improved sample preparation and data analysis

“…Various methods have been applied in recent years for the discovery of biomarkers or biomarker patterns of major human diseases, especially for various types of cancer [1][2][3][4][5][6][7][8][9][10][11][12][13]. Amongst these surface enhanced laser desorption ionization mass spectrometry (SELDI-MS), which combines on-chip sample preparation with mass spectrometric analysis, has taken a prominent position [14], although more recent results question the viability of this approach [15,16].…”

Analysis of human serum phosphopeptidome by a focused database searching strategy

“…Based on this assumption, the parent protein sequences for these endogenous nonphosphopeptides could form a small database for serum phosphopeptidome analysis. To this aim, all the serum proteins identified by peptidome analysis in literature including that identified by Hu et al [21] in our group and by several other labs [1][2][3][4][5] were collected. Totally 850 proteins were obtained.…”

“…The human serum peptidome, which contains thousands of endogenous peptides either degraded from proteins or secreted from cells and tissues, has been extensively investigated [1][2][3][4][5]. It is believed that the studies on human serum peptidome hold great promise for finding potential biomarkers of serious diseases such as cancers [6].…”

Peptidomic analysis of breast cancer reveals a putative surrogate marker for estrogen receptor-negative carcinomas

“…The methodology has been described by Schulte et al, 8 Tammen et al, 9 and Rai et al 10 in detail. Briefly, peptides are extracted from biological sources and fractionated by means of reversed phase chromatography.…”