LC-MS/MS Quantification of Zn- 2 Glycoprotein: A Potential Serum Biomarker for Prostate Cancer

Background: Zn2 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. Methods: We selected the ZAG tryptic peptide EIPAWVPEDPAAQITK 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. Results: 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]. Conclusions: This LC-MS/MS assay is reproducible and can be used to evaluate the clinical utility of ZAG as a cancer biomarker. © 2007 American Association for Clinical Chemistry Zn2-glycoprotein (ZAG) is a glycoprotein with a molecular mass of 41 000 Da and a crystal structure similar to that of a class I major histocompatibility complex (1, 2). Biochemically, ZAG stimulates lipid degeneration in adipocytes and appears to be involved in cachexia, a wasting syndrome that can affect people with cancer, AIDS, and other terminal illnesses (3, 4). ZAG appears naturally in most body fluids, such as blood (5 ), sweat (6 ), seminal fluid (7 ), breast cyst fluid (8 ), cerebrospinal fluid (9 ), and urine (10 ) and is also found in secretory epithelial cells of the liver and the gastrointestinal tract (11 ). Previous studies employing techniques such as immunohistology and 2-dimensional electrophoresis have reported that ZAG is overexpressed in certain malignant tumors and thus may serve as a potential cancer biomarker (12, 13). ZAG quantification in serum by immunoassay found circulating concentrations ranging from 40 mg/L in healthy individuals to 120 mg/L in some diseased persons (14 ). In this study we used liquid chromatography–tandem mass spectrometry (LC-MS/MS), at a high LC flow rate commonly used in the clinical laboratory (250 L/min), combined with proteolysis, to quantify ZAG in serum. We also determined whether ZAG could be used as a specific biomarker for prostate cancer (PCa).