A functional annotation of subproteomes in human plasma.

The data collected by Human Proteome Organization's Plasma Proteome Pilot project phase was analyzed by members of our working group. Accordingly, a functional annotation of the human plasma proteome was carried out. Here, we report the findings of our analyses. First, bioinformatic analyses were undertaken to determine the likely sources of plasma proteins and to develop a protein interaction network of proteins identified in this project. Second, annotation of these proteins was performed in the context of functional subproteomes involved in the coagulation pathway, the mononuclear phagocytic system, the inflammation pathway, the cardiovascular system, and the liver; as well as the subset of proteins associated with DNA binding activities. Our analyses contributed to the Plasma Proteome Database (http://www.plasmaproteomedatabase.org), an annotated database of plasma proteins identified by HPPP as well as from other published studies. In addition, we address several methodological considerations including the selective enrichment of post-translationally modified proteins by the use of multi-lectin chromatography as well as the use of peptidomic techniques to characterize the low molecular weight proteins in plasma. Furthermore, we have performed additional analyses of peptide identification data to annotate cleavage of signal peptides, sites of intra-membrane proteolysis and post-translational modifications. The HPPP-organized, multi-laboratory effort, as described herein, resulted in much synergy and was essential to the success of this project.

Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: systematic variation between sample types and calibration of mass spectrometry data.

Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets using the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody-based methods will further advance the scientific goals of the PPP.

Distinct proteome features of plasma microparticles.

Plasma microparticles (MPs) are spherical cell membrane fragments derived from either apoptotic or activated cells. Characterized by a rich phospholipid moiety and many protein constituents, MPs normally circulate in the blood and contribute to numerous physiological processes. In disease states, MPs derived from the injured organ likely contain valuable markers for determining the site, type, and extent of disease pathology. However, the basic protein characteristics of plasma MPs have yet to be described. In this study, MPs from a pooled plasma sample derived from 16 healthy donors, all of group A blood type, were prepared by ultracentrifugation. Flow cytometry confirmed that a majority of these MPs are smaller than 1 microm. Factor Xa generation assay revealed the presence of tissue factor activity in these MPs, confirming MPs' role in initiating blood coagulation. The MP proteome was analyzed by two-dimensional (2-D) gel electrophoresis performed in triplicate, and compared with a 2-D gel of pooled whole plasma and blood platelets. Overall, plasma MPs displayed distinct protein features and a greater number of protein spots (1021-1055) than that detected in whole plasma (331-370). Protein spots expressed in high abundance in the MP proteome were then excised and submitted for protein identity determination. This process provided protein identification for 169 protein spots and reported their relative protein quantities within the MP proteome. These 169 protein spots represented 83 different proteins and their respective isoforms. Thirty of these proteins have never before been reported in previous proteome analyses of human plasma. These results provide unprecedented information on the MP proteome and create a basis for future studies to understand MP biology and pathophysiology.