Plasma proteins represent an important part of the human proteome. Although recent proteomics research efforts focus largely on determining the overall number of proteins circulating in plasma, it is equally important to delineate protein variations among individuals, because they can signal the onset of diseases and be used as biological markers in diagnostics. To date, there has been no systematic proteomics effort to characterize the breadth of structural modifications in individual proteins in the general population. In this work, we have undertaken a population proteomics study to define gene-and protein-level diversity that is encountered in the general population. Twenty-five plasma proteins from a cohort of 96 healthy individuals were investigated through affinity-based mass spectrometric assays. A total of 76 structural forms͞variants were observed for the 25 proteins within the samples cohort. Posttranslational modifications were detected in 18 proteins, and point mutations were observed in 4 proteins. The frequency of occurrence of these variations was wide-ranged, with some modifications being observed in only one sample, and others detected in all 96 samples. Even though a relatively small cohort of individuals was investigated, the results from this study illustrate the extent of protein diversity in the human population and can be of immediate aid in clinical proteomics͞biomarker studies by laying a basal-level statistical foundation from which protein diversity relating to disease can be evaluated.mass spectrometry ͉ population proteomics ͉ protein modifications

Background-The myocardium secretes B-type natriuretic peptide (BNP) in response to stimuli associated with heart failure (HF). However, high immunoreactive-BNP levels in patients with HF are associated with a paradoxical lack of natriuretic response. We hypothesized that commercially available assays for immunoreactive BNP do not reflect the bioactivity of the natriuretic peptide system, because they measure both unprocessed inactive pro-BNP and mature BNP 1-32. We describe an assay for the detection of bioactive BNP 1-32 and confirm very low concentrations in plasma from HF patients. Methods and Results-We developed a quantitative mass spectrometry immunoassay to capture endogenous BNP peptides using high affinity antibodies. Bound BNP and its truncated fragments were detected by matrix assisted laser desorption ionization-time of flight mass spectrometry based on their predicted masses. Mass spectrometry immunoassay revealed rapid in vitro degradation of BNP 1-32 in plasma, which requires plasma collection in the presence of high protease inhibitor concentrations. In 11 of 12 HF patients BNP 1-32 was detectable, ranging from 25 to 43 pg/mL. Several degraded forms of BNP were also detected at similarly low levels. In contrast, parallel measurements of immunoreactive BNP using the Biosite assay ranged from 900 to 5000 pg/mL. Conclusions-Detection of endogenous BNP 1-32 requires special preservation of plasma samples. Mass spectrometry immunoassay technology demonstrates that HF patients have low levels of BNP 1-32. Commercially available immunoreactive-BNP assays overrepresent biological activity of the natriuretic peptide system because they cannot distinguish between active and inactive forms. This observation may, in part, explain the "natriuretic paradox." (Circ Heart Fail. 2008;1:258-264.)

A high-throughput mass spectrometric immunoassay (MSIA) system for the analysis of proteins directly from biological fluids is reported. A 96-well-format robotic workstation equipped with antibody-derivatized affinity pipet tips was used for the parallel extraction of specific proteins from samples and subsequent deposition onto 96-well arrayed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) targets. Interferences from nonspecifically bound proteins were minimized through choice of appropriate affinity pipet tip derivatization chemistries. Sample preparation for MALDI-TOFMS was enhanced through the use of hydrophobic/hydrophilic contrasting targets, which also presented functionalities found to promote matrix/analyte crystal growth. Automated mass spectrometry was used in the unattended acquisition of data, resulting in an analysis rate of approximately 100 samples/h (biological fluid-->data). The quantitative MSIA of beta2m levels present in human plasma samples is given as illustration.