Human Serum Proteins Preseparated by Electrophoresis or Chromatography Followed by Tandem Mass Spectrometry

Abstract

Electrophoretic and chromatographic sample preparations were compared and together detected the presence of some 600 types of protein products in human serum. Proteins from crude serum preseparated by ionic electrophoresis, chromatography, or a combination of both were analyzed. Proteins were digested with trypsin or chymotrypsin. Naturally occurring peptides were also collected by reversed-phase chromatography. The resulting peptides were identified by tandem mass spectrometry. The peptides were either desorbed by a laser from a metal chip into a quadrupole-time-of-flight mass spectrometer or ionized as an electro-spray from reversed-phase chromatography via a metal needle under voltage into an ion-trap mass spectrometer. All of the commonly known proteins associated with serum were detected, and the two mass spectrometers agreed on the identity of abundant serum proteins. Preseparation of serum proteins prior to digestion markedly enhanced the capacity to detect un-common proteins from blood. Electrophoretic- and chromatography-based experiments were found to be complementary. Many novel cellular proteins not previously associated with serum were recorded.

Identification of Candidate Biomarker Proteins Released by Human Endometrial and Cervical Cancer Cells Using Two-Dimensional Liquid Chromatography/Tandem Mass Spectrometry

“…Significantly, AAT is a wellestablished abundant serum protein; 29 in addition, other independent, large-scale studies on serum have reported the presence of many of the proteins listed in Table 1. 21,[30][31][32][33][34] Other identified proteins of note are also listed in Table 1. Details of peptides identified are given in Supplementary Table 5s in Supporting Information.…”

Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly‐available database

“…Like Aebersold and Mann [2], they conclude that no single analytical approach will identify all the major proteins in any proteome. Others have recently used similar 2-D separation of peptides offline, intact protein fractionation prior to MS, or sensitive ESI-MS/MS analysis of fractionated peptides [36][37][38][39]. As far as cost-effectiveness, the 2-D HPLC approach required much more time and labor and was much less suited to automation than the other strategies; it has the advantage of being able to process large volumes of sample, when that is available and desired.…”

“…To test the feasibility of this approach, we searched all ORFs using peak list data from six PPP laboratories (17,30,37,41,52,55). NCBI human genome sequence build 33 was translated in all three reading frames and both strands; all non-redundant ORFs were assembled into chromosome specific sequence collections.…”

A proteomic study of the HUPO Plasma Proteome Project's pilot samples using an accurate mass and time tag strategy

“…One school of thought has been that nearly all peptides result from highly specific digestion by the exogenous protease, typically trypsin, and thus all confident peptide identifications should conform to fully tryptic digestion patterns (e.g., [10,48]). Alternatively, complex protein mixtures processed by endogenous proteases may contain unexpected amino-and carboxy-termini, resulting in non-tryptic cleavage states (e.g., [15,18,27]).…”

“…The Plasma Proteomics Initiative is timely, as blood plasma and serum are widely recognized as body fluids of great promise for human health for diagnostics, e.g., disease prognostics and clinical monitoring [2,4,[6][7][8][9][10][11][12][13][14][15][16][17][18]. Two of the most compelling reasons for studying human plasma are 1) the universal availability of sufficient blood plasma and serum for method development and validation and 2) the long-standing use of plasma and serum as a source of clinically relevant information [6,19].…”