Antibody-based analytical methods can provide quantitative, reproducible, and sensitive measurements of specific analytes. These capabilities are valuable both for routine clinical analysis and for the high-throughput exploration of hypotheses regarding specific proteins. The multiplexing of antibody-based assays through the use of planar microarrays [1-6] and bead arrays has opened up new research opportunities. The various formats of antibody microarrays and the applications and relative merits of each are reviewed elsewhere . Several goals of the Plasma Proteome Project (PPP) of the HUPO can be advanced through the use of antibody-based methods.
One of the major goals of the pilot phase of the PPP was to determine the effects of the blood preparation method on the quality of proteomic data. Blood may be prepared as serum (the soluble portion of clotted blood) or as plasma (the soluble portion of anticoagulated blood), and various anticoagulants may be used to make plasma. Before attempting a large-scale study of the human plasma proteome, it is necessary to determine ifthe preparation method introduces systematic alterations to the levels of all proteins or specific proteins, or whether certain preparation methods are desirable or not for certain applications. Antibody-based methods are well suited to study that question, since the levels of multiple proteins may be precisely and accurately measured in multiple samples. An additional valuable use of antibody-based methods for the PPP is to provide complementary information to the broad-based discovery capabilities of separations and MS methods.
The exploration of these topics was facilitated by the assembly of human serum and plasma reference specimens by BD Diagnostics (Franklin Lakes, NJ), the National Institute for Biological Standards and Control (NIBSC, UK), and the Chinese Academy of Medical Sciences (CAMS, Beijing) . Blood samples, each pooled from a male and female donor, were prepared in four ways: as serum, as plasma anticoagulated with sodium citrate, as plasma anticoagulated with K-EDTA, and as plasma anticoagulated with lithium heparin. Four different laboratories used antibody-based methods to analyze the reference specimens, with each laboratory using a distinct method. The combined data sets were used to investigate the level of systematic variation in protein levels introduced by the preparation methods and to gain further insight into the suitability of the various methods for proteomic analyses. We evaluated the following: evidence for bias in the concentrations of all the proteins in general; evidence for protein-specific alterations in concentration as a function of preparation method; and the relationship between the detectability of proteins by MS and their concentrations in plasma or serum.
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