Sjöström Martin, Ossola Reto, Breslin Thomas, Rinner Oliver, Malmström Lars, Schmidt Alexander, Aebersold Ruedi, Malmström Johan, Niméus Emma
§Biognosys AG, 8952 Schlieren, Switzerland.
#Biozentrum, University of Basel, 4003 Basel, Switzerland.
J Proteome Res. 2015 Jul 2;14(7):2807-18. doi: 10.1021/acs.jproteome.5b00315. Epub 2015 Jun 5.
It is of highest importance to find proteins responsible for breast cancer dissemination, for use as biomarkers or treatment targets. We established and performed a combined nontargeted LC-MS/MS and a targeted LC-SRM workflow for discovery and validation of protein biomarkers. Eighty breast tumors, stratified for estrogen receptor status and development of distant recurrence (DR ± ), were collected. After enrichment of N-glycosylated peptides, label-free LC-MS/MS was performed on each individual tumor in triplicate. In total, 1515 glycopeptides from 778 proteins were identified and used to create a map of the breast cancer N-glycosylated proteome. Based on this specific proteome map, we constructed a 92-plex targeted label-free LC-SRM panel. These proteins were quantified across samples by LC-SRM, resulting in 10 proteins consistently differentially regulated between DR+/DR- tumors. Five proteins were further validated in a separate cohort as prognostic biomarkers at the gene expression level. We also compared the LC-SRM results to clinically reported HER2 status, demonstrating its clinical accuracy. In conclusion, we demonstrate a combined mass spectrometry strategy, at large scale on clinical samples, leading to the identification and validation of five proteins as potential biomarkers for breast cancer recurrence. All MS data are available via ProteomeXchange and PASSEL with identifiers PXD001685 and PASS00643.
寻找导致乳腺癌扩散的蛋白质作为生物标志物或治疗靶点至关重要。我们建立并执行了一种结合非靶向液相色谱 - 串联质谱（LC-MS/MS）和靶向液相色谱 - 选择反应监测（LC-SRM）的工作流程，用于蛋白质生物标志物的发现和验证。收集了80例乳腺肿瘤，根据雌激素受体状态和远处复发情况（DR ±）进行分层。在富集N-糖基化肽后，对每个肿瘤个体进行三次无标记的LC-MS/MS分析。总共鉴定出778种蛋白质的1515种糖肽，并用于创建乳腺癌N-糖基化蛋白质组图谱。基于此特定蛋白质组图谱，我们构建了一个92重靶向无标记LC-SRM分析板。通过LC-SRM对这些蛋白质在各个样本中进行定量，结果显示在DR +/DR -肿瘤之间有10种蛋白质始终存在差异调节。在一个独立队列中，对5种蛋白质在基因表达水平上作为预后生物标志物进行了进一步验证。我们还将LC-SRM结果与临床报告的HER2状态进行了比较，证明了其临床准确性。总之，我们展示了一种在临床样本上大规模应用的组合质谱策略，从而鉴定并验证了5种蛋白质作为乳腺癌复发的潜在生物标志物。所有质谱数据可通过ProteomeXchange和PASSEL获取，标识符分别为PXD001685和PASS00643。
