Identification of a novel proteoform of prostate specific antigen (SNP-L132I) in clinical samples by multiple reaction monitoring

Abstract

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.

Fig. 1.

Detection of three possible combinations of allele expressions in examples of analyses in clinical samples. Endogenous signals of LSEPAELTDAVK and LSEPAEITDAVK are shown in red, and their corresponding heavy-labeled IS signals are in blue.

Fig. 2.

Correlations between the measured concentrations of PSA peptides in (A–C) seminal and (D–F) blood plasma samples.

Fig. 3.

Correlation between the PSA levels determined via MRM and DELFIA^® assays in (A–C) seminal and (D–F) blood plasma samples.

Fig. 4.

Linearity of the MRM assay determined by using heavy-labeled IS peptides spiked into a pooled blood plasma sample at various concentrations (0.03–30 fmol/μl). The measured levels of the corresponding endogenous peptides are represented by blue diamonds, and the limit of quantification (cv < 20%) is indicated with arrow.