Toward a Human Blood Serum Proteome

Abstract

Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.Molecular & Cellular Proteomics 1:947-955, 2002.

Recent advances in blood‐related proteomics

“…It is therefore of importance to have good preanalytical conditions when mining the proteome of plasma, and more particularly its "peptidome", in which about 5000 peptides have been revealed [22]. Over 10 000 different proteins have been estimated to be commonly present in the plasma, most of which are at very low relative abundances [23]. A major challenge of proteomic analysis of plasma is the low abundance proteins, like (1) proteins or peptides resulting from tissue leakage, (2) proteins released from normal cells as a result of cell death or damage, (3) proteins released from tumor cells as aberrant secretions.…”

Building towards a standardised approach to biocorrosion studies: a review of factors influencing Mg corrosion in vitro pertinent to in vivo corrosion

“…Proteins, in particular albumin, are an essential competent of human plasma [79] and are the majority component of plasma aside from water, as shown in Table 1. A number of studies have considered the influence proteins have on the biocorrosion of Mg alloys.…”

“…Amino acids in particular have gathered interest in biocorrosion studies [76], presumably because of their presence in certain SBFs such as MEM as shown in Table 1. These additional components, such as amino acids, are at much lower concentrations in blood compared to proteins [79,89].…”

Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

“…Such comprehensive analyses of human serum proteomes are complicated due to their content of thousands of different proteins, which are present in a wide range of concentrations [3][4][5][6][7][8][9][10]. The first phase of the PPP project, described in different articles in this journal, aims at identifying as many proteins as possible from tested samples of human sera or plasma that were distributed by the PPP to participating laboratories.…”

“…This would result in less interference between peptides during the LC-MS/MS, thus leading to larger numbers of identified peptides. Very large increases in the number of identified peptides and proteins in complex samples were recently achieved by using multidimensional protein identification technology (MudPIT) combining strong cation exchange (SCX) with long RP gradients [3,6,11] (reviewed in [12][13][14]) and by performing repeated chromatographies with mass segmentation selection of peptides for fragmentation [3,6]. However, even with the increased number of steps employed for prefractionation, the number of identified proteins eventually reaches a plateau, since the concentration of some rare proteins is still below the needed threshold level of sensitivity for the mass spectrometers.…”