LC-MS/MS Quantification of Zn-α2 Glycoprotein: A Potential Serum Biomarker for Prostate Cancer

Abstract

Zn-alpha2 glycoprotein (ZAG) is a relatively abundant glycoprotein that has potential as a biomarker for prostate cancer. We present a high-flow liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring serum ZAG concentrations by proteolytic cleavage of the protein and quantification of a unique peptide. We selected the ZAG tryptic peptide (147)EIPAWVPEDPAAQITK(162) as the intact protein for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Standards using recombinant ZAG in bovine serum albumin, 50 g/L, and a pilot series of patient sera were denatured, reduced, alkylated, and digested with trypsin. The concentration of ZAG was calculated from a dose-response curve of the ratio of the relative abundance of the ZAG tryptic peptide to internal standard. The limit of detection for ZAG in serum was 0.08 mg/L, and the limit of quantification was 0.32 mg/L with a linear dynamic range of 0.32 to 10.2 mg/L. Replicate digests from pooled sera run during a period of 3 consecutive days showed intraassay imprecision (CV) of 5.0% to 6.3% and interassay imprecision of 4.4% to 5.9%. Mean (SD) ZAG was higher in 25 men with prostate cancer [7.59 (2.45) mg/L] than in 20 men with nonmalignant prostate disease [6.21 (1.65) mg/L, P = 0.037] and 6 healthy men [3.65 (0.71) mg/L, P = 0.0007]. This LC-MS/MS assay is reproducible and can be used to evaluate the clinical utility of ZAG as a cancer biomarker.