Shi Tujin, Gao Yuqian, Quek Sue Ing, Fillmore Thomas L, Nicora Carrie D, Su Dian, Zhao Rui, Kagan Jacob, Srivastava Sudhir, Rodland Karin D, Liu Tao, Smith Richard D, Chan Daniel W, Camp David G, Liu Alvin Y, Qian Wei-Jun
Biological Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99352.
J Proteome Res. 2014 Feb 7;13(2):875-82. doi: 10.1021/pr400912c. Epub 2013 Dec 3.
Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase liquid chromatography (LC) separations to fractionate and select target fractions for follow-on LC-selected reaction monitoring (LC-SRM) analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and limit of quantification (LOQ) values of ∼130 pg/mL in serum and ∼10 pg per 100 μg of total protein mass in urine, respectively. A good correlation (R(2) = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and enzyme-linked immunosorbent assay (ELISA). On the basis of an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between noncancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.
前梯度2（AGR2）是多种上皮癌细胞中一种与癌症相关的分泌蛋白。我们开发了一种高灵敏度的靶向质谱分析法，用于定量尿液和血清中的AGR2。临床样本中的消化肽通过PRISM（高压和高分辨率分离与智能选择和多重分析相结合）进行处理，该方法采用高pH反相液相色谱（LC）分离来分馏和选择目标馏分，用于后续的LC选择反应监测（LC-SRM）分析。AGR2的PRISM-SRM分析显示，血清中的变异系数（CV）<10%，定量限（LOQ）值约为130 pg/mL，尿液中每100 μg总蛋白质量约为10 pg。SRM和酶联免疫吸附测定（ELISA）之间尿液中可测量的AGR2浓度具有良好的相关性（R² = 0.91）。基于37名受试者的初始队列，非癌症组和癌症组之间尿液AGR2/PSA浓度比存在显著差异（P = 0.026）。需要进行大型临床队列研究来验证AGR2作为前列腺癌有用诊断生物标志物的有效性。我们的工作验证了通过基因组学鉴定候选分泌蛋白生物标志物并通过靶向蛋白质组学进行测量的方法，特别是对于没有免疫测定方法的蛋白质。
