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Recently, the number of mass spectrometry-based quantification assays has been increased, partially due to the global efforts of chromosome-centric human proteome project (C-HPP). Our goal at the Chromosome 19 Consortium is to provide novel selected reaction monitoring (SRM) assays of proteins coded on chromosome 19. We have selected the two most useful signature peptides (NSQVWLGR and HNLFEPEDTGQR) of human kallikrein-2 (hK2 – NX_P20151) and developed an SRM assay. Details of the analytical parameters, including multiple transitions by peptides, are presented. The endogenous levels of hK2 were determined in clinical samples (n= 35). The limit of quantification was also estimated by spiking heavy isotope-labeled peptides into seminal plasma samples at various concentrations (LOQ ≈ 29 ng/mL).

A multiple reaction monitoring (MRM) assay was developed for precise quantitation of 87 plasma proteins including the three isoforms of apolipoprotein E (APOE) associated with cardiovascular diseases using nanoscale liquid chromatography separation and stable isotope dilution strategy. The analytical performance of the assay was evaluated and we found an average technical variation of 4.7% in 4–5 orders of magnitude dynamic range (≈0.2 mg/L to 4.5 g/L) from whole plasma digest. Here, we report a complete workflow, including sample processing adapted to 96-well plate format and normalization strategy for large-scale studies. To further investigate the MS-based quantitation the amount of six selected proteins was measured by routinely used clinical chemistry assays as well and the two methods showed excellent correlation with high significance (p-value < 10e-5) for the six proteins, in addition for the cardiovascular predictor factor, APOB: APOA1 ratio (r = 0.969, p-value < 10e-5). Moreover, we utilized the developed assay for screening of biobank samples from patients with myocardial infarction and performed the comparative analysis of patient groups with STEMI (ST- segment elevation myocardial infarction), NSTEMI (non ST- segment elevation myocardial infarction) and type-2 AMI (type-2 myocardial infarction) patients.

Prostate specific antigen (PSA) is a well-established tumor marker that is frequently employed as model biomarker in the development and evaluation of emerging quantitative proteomics techniques, partially as a result of wide access to commercialized immunoassays serving as “gold standards.” We designed a multiple reaction monitoring (MRM) assay to detect PSA proteoforms in clinical samples (n= 72), utilizing the specificity and sensitivity of the method. We report, for the first time, a PSA proteoform coded by SNP-L132I (rs2003783) that was observed in nine samples in both heterozygous (n= 7) and homozygous (n= 2) expression profiles. Other isoforms of PSA, derived from protein databases, were not identified by four unique proteotypic tryptic peptides. We have also utilized our MRM assay for precise quantitative analysis of PSA concentrations in both seminal and blood plasma samples. The analytical performance was evaluated, and close agreement was noted between quantitations based on three selected peptides (LSEPAELTDAVK, IVGGWECEK, and SVILLGR) and a routinely used commercialized immunoassay. Additionally, we disclose that the peptide IVGGWECEK is shared with kallikrein-related peptidase 2 and therefore is not unique for PSA. Thus, we propose the use of another tryptic sequence (SVILLGR) for accurate MRM quantification of PSA in clinical samples.

Cigarette smoking results in variable degrees of inflammation in the lower respiratory tract. Furthermore, smoking produces oxidant-mediated changes in the lung, important to the pathogenesis of emphysema. Since glutathione can neutralize reactive oxygen species and prevent peroxidation of unsaturated lipids, it may constitute an important component of the lung's defense against oxidant and inflammatory injury. In the present study, broncholaveolar lavage (BAL) was performed in 27 smokers, and the concentrations of total glutathione as well as the cellular and humoral markers of inflammatory activity were studied. There were significant correlations between total glutathione and neutrophils; two neutrophil granule components, myeloperoxidase and elastase; and chemotactic activity for neutrophils. Moreover, the total glutathione correlated with the eosinophil cationic protein (ECP), a granule constituent of the eosinophil, with two locally produced antiproteases, secretory leukocyte protease inhibitor (SLPI) and antichymotrypsin (ACHY), but not with an a1-protease inhibitor and albumin. These data suggest that the total glutathione levels in BAL fluid may reflect a degree of oxidative and inflammatory stress caused by cigarette smoke, and they are therefore likely to contribute to the protection against this stress.