Quantitative pattern analysis of the N‐terminally processed isoforms of platelet factor‐4 in serum

RATIONALE Platelet factor 4 (PF4) is a small cytokine belonging to the CXC chemokine family which has been shown to play a role in inflammation and in the regulation of angiogenesis. In general, chemokines are susceptible to proteolytic cleavage in amino and carboxy terminal regions, which usually results in dramatic changes to the chemokine bioactivity. The purpose of this study was to identify various platelet factor‐4 (PF4) isoforms caused by proteolytic processing and to quantify their levels in normal serum. METHODS First, we identified the N‐terminally truncated PF4 isoforms from a standard purified PF4 protein sample by using mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. Then, we used high‐performance liquid chromatography (HPLC) to semi‐purify PF4 from serum samples, and the levels of the four most abundant PF4 isoforms were quantitatively determined using selected reaction monitoring (SRM) assays on a nano‐LC/triple‐quadrupole mass spectrometer. RESULTS We have identified seven N‐terminally processed PF4 isoforms and compared the levels of major PF4 isoforms from nine serum samples. Pro‐p1 (EAEEDGDLQCLCVK–; average MW, 7765.2) is the major PF4 isoform in serum whereas the PF4 isoforms, designated Prot‐p4 (FASAEAEEDGDLQCLCVK– ;average MW, 8141.5), Prot‐p3 (SAEAEEDGDLQCLCVK– ; average MW, 7923.3), and Prot‐p2 (AEEDGDLQCLCVK– ; average MW, 7836.3), are at about 16%, 3%, and 2% levels of the major one, respectively. CONCLUSIONS This study is the first report on the levels of N‐terminally processed PF4 isoforms in serum. Also, this study shows the usefulness of SRM in determining concentrations of protein isoform variants, which can be often overlooked in immunoassay analysis. Copyright © 2013 John Wiley & Sons, Ltd.