The human serum proteome: Display of nearly 3700 chromatographically separated protein spots on two‐dimensional electrophoresis gels and identification of 325 distinct proteins

Abstract

Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.

Involvement of proteases in porcine reproductive and respiratory syndrome virus uncoating upon internalization in primary macrophages

“…1-antichymotrypsin, 1-antitrypsin, 1-macroglobulin, 2-macroglobulin, antiplasmin, antithrombin III, ceruloplasmin, haptoglobins and heparin cofactor II [1,32]. To investigate if the antiproteolytic property of -globulins caused the inhibition of PRRSV infection, macrophages were treated with a commercial broad-spectrum protease inhibitor cocktail (Complete™ EDTA-free protease inhibitor cocktail) at a concentration recommended by the manufacturer.…”

A path or a new road in laboratory diagnostics? Biological mass spectrometry: Facts and perspectives

“…However, the examination of the amino acid structure has revealed that these are not all distinct molecules: more than one third of them are the results of post-translational (epigenetic) modifications. 24 The maximum number of distinct (non-redundant) serum molecules is now estimated to be 1,175. 27 One of the most interesting results of these examinations is that the proteins in highest number are not really serum proteins, rather cell-originated, membrane, cytoplasmic, cytoskeletal, endoplasmic reticular, or lysosomal proteins, which are present in minute quantities but may be important from the view of diagnosis.…”

“…Various mass spectrometric techniques are again of crucial importance in this work, because they are capable of carrying out the job, 20 as this is the area from where they came into clinical diagnostic use. If we know the molar mass of a peptide, we can turn to partly already existing international databases, 24,28 which register data for macromolecules of human origin. In the future we can also rely on further international databases that are developed in order to make practical use of proteomics.…”

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.…”