Department of Biochemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6350, USA.
J Proteome Res. 2012 Jul 6;11(7):3908-13. doi: 10.1021/pr300161j. Epub 2012 Jun 26.
Tumor-derived mutant KRAS (v-Ki-ras-2 Kirsten rat sarcoma viral oncogene) oncoprotein is a critical driver of cancer phenotypes and a potential biomarker for many epithelial cancers. Targeted mass spectrometry analysis by multiple reaction monitoring (MRM) enables selective detection and quantitation of wild-type and mutant KRAS proteins in complex biological samples. A recently described immunoprecipitation approach (Proc. Nat. Acad. Sci.2011, 108, 2444-2449) can be used to enrich KRAS for MRM analysis, but requires large protein inputs (2-4 mg). Here, we describe sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based enrichment of KRAS in a low molecular weight (20-25 kDa) protein fraction prior to MRM analysis (GeLC-MRM). This approach reduces background proteome complexity, thus, allowing mutant KRAS to be reliably quantified in low protein inputs (5-50 μg). GeLC-MRM detected KRAS mutant variants (G12D, G13D, G12V, G12S) in a panel of cancer cell lines. GeLC-MRM analysis of wild-type and mutant was linear with respect to protein input and showed low variability across process replicates (CV = 14%). Concomitant analysis of a peptide from the highly similar HRAS and NRAS proteins enabled correction of KRAS-targeted measurements for contributions from these other proteins. KRAS peptides were also quantified in fluid from benign pancreatic cysts and pancreatic cancers at concentrations from 0.08 to 1.1 fmol/μg protein. GeLC-MRM provides a robust, sensitive approach to quantitation of mutant proteins in complex biological samples.
肿瘤衍生的突变 KRAS（v-Ki-ras-2 Kirsten 大鼠肉瘤病毒癌基因）癌蛋白是癌症表型的关键驱动因素，也是许多上皮癌的潜在生物标志物。通过多重反应监测（MRM）进行的靶向质谱分析能够选择性地检测和定量复杂生物样品中的野生型和突变 KRAS 蛋白。最近描述的一种免疫沉淀方法（Proc. Nat. Acad. Sci.2011, 108, 2444-2449）可用于富集 KRAS 进行 MRM 分析，但需要大量的蛋白质输入（2-4 mg）。在这里，我们描述了在进行 MRM 分析（GeLC-MRM）之前，基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的 KRAS 在低分子量（20-25 kDa）蛋白质馏分中的富集。这种方法减少了背景蛋白质组的复杂性，从而能够在低蛋白输入（5-50 μg）下可靠地定量突变 KRAS。GeLC-MRM 在一系列癌细胞系中检测到 KRAS 突变变体（G12D、G13D、G12V、G12S）。野生型和突变型的 GeLC-MRM 分析与蛋白质输入呈线性关系，并且在过程重复之间显示出低变异性（CV = 14%）。来自高度相似的 HRAS 和 NRAS 蛋白的肽的同时分析使能够对这些其他蛋白对 KRAS 靶向测量的贡献进行校正。KRAS 肽在良性胰腺囊肿和胰腺癌的液体中也以 0.08 至 1.1 fmol/μg 蛋白的浓度进行定量。GeLC-MRM 为复杂生物样品中突变蛋白的定量提供了一种强大、灵敏的方法。
