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

Abstract

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.

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

“…Four different immunoassay and antibody microarray methods were performed by four independent laboratories (DadeBehring, Genomics Institute of Novartis Foundation, Molecular Staging, and Van Andel Research Institute). A total of 323 assays measured 237 unique analytes (Haab et al [22]). In the cases of multiple assays, we cannot be certain that the same epitopes were targeted.…”

“…After extensive curation, we matched 76 IPI proteins among the 9504 dataset (based on one or more peptides) and 49 proteins among the 3020 protein dataset (based on two or more peptides) to quantitative analytes. Figure 1 in Haab et al [22] shows four parameters used to determine the sensitivity of detection of these proteins as a function of immunoreactive concentration: number of labs reporting that protein, number of peptides on which protein IDs were based, percent coverage of the protein sequence, and score. The correlation coefficient for the total number of peptides matching that protein is r = 0.86 for the 3020 dataset and r = 0.90 for the 9504 protein dataset: log10(N) = 0.365*log10(conc)20.711.…”

“…Haab et al [22] extensively analyzed the concentrations of assayable proteins in the PPP specimen sets. They noted a systematic 15% lower value for many proteins in citrateplasma, compared with other specimens; it turns out that this can be attributed to dilution and osmotic effect with the citrate solution, without any impairment in detection of proteins compared with the other specimens.…”

Human body fluid proteome analysis

“…The proteomics platforms tested included LC-FT-ICR MS [50], antibody array [51] [42], and SELDI-TOF-MS [54] (Table 1). An important observation was that different platforms revealed a distinct subset of proteins and were more likely complementary toward identification of serum proteins [52].…”

Standards for Plasma and Serum Proteomics in Early Cancer Detection: A Needs Assessment Report from the National Institute of Standards and Technology-National Cancer Institute Standards, Methods, Assays, Reagents and Technologies Workshop, August 18–19, 2005

“…EDTA plasma was recommended as the preferred standard specimen, because with its considerable variability, plasma avoids the additional time ex vivo for clotting, and EDTA gave more consistent results with less proteolytic cleavage than heparin-or citrate-anticoagulated plasma (11,15,24 ). For particular analytes, other choices may be indicated.…”