Bondar Olga P, Barnidge David R, Klee Eric W, Davis Brian J, Klee George G
Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA.
Clin Chem. 2007 Apr;53(4):673-8. doi: 10.1373/clinchem.2006.079681. Epub 2007 Feb 22.
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.
锌α2糖蛋白（ZAG）是一种相对丰富的糖蛋白，具有作为前列腺癌生物标志物的潜力。我们提出了一种高流量液相色谱 - 串联质谱（LC-MS/MS）方法，通过蛋白质的蛋白水解裂解和对一种独特肽段的定量来测量血清ZAG浓度。
我们选择ZAG胰蛋白酶肽段（147）EIPAWVPEDPAAQITK（162）作为完整蛋白进行定量，并使用具有该序列的稳定同位素标记合成肽作为内标。使用重组ZAG在50 g/L牛血清白蛋白中的标准品以及一系列患者血清的预实验样本进行变性、还原、烷基化处理，并用胰蛋白酶消化。ZAG浓度根据ZAG胰蛋白酶肽段与内标的相对丰度比值的剂量反应曲线计算得出。
血清中ZAG的检测限为0.08 mg/L，定量限为0.32 mg/L，线性动态范围为0.32至10.2 mg/L。连续3天对混合血清进行重复消化显示，批内不精密度（CV）为5.0%至6.3%，批间不精密度为4.4%至5.9%。25名前列腺癌男性患者的ZAG平均（SD）值[7.59（2.45）mg/L]高于患有非恶性前列腺疾病的20名男性患者[6.21（1.65）mg/L，P = 0.037]和6名健康男性[3.65（0.71）mg/L，P = 0.0007]。
这种LC-MS/MS检测方法具有可重复性，可用于评估ZAG作为癌症生物标志物的临床效用。
