Department of Neurology, Erasmus MC, Rotterdam, The Netherlands.
J Proteome Res. 2011 Jan 7;10(1):288-98. doi: 10.1021/pr100709b. Epub 2010 Dec 3.
Barrett's esophagus (BE) is associated with increased risk of esophageal adenocarcinoma (EAC) and characterized by replacement of normal esophageal squamous epithelium by columnar epithelium. These alterations are also reflected in changes in the protein-expression profiles of the cell types involved. To separately investigate the proteomes of selected cell-types we combined laser-capture microdissection (LCM) and liquid chromatography-mass spectrometry (LC-MS). Aims were to determine the sensitivity, specificity, and technical reproducibility of the sampling method, and the biological variability within and between biopsies and patients. Frozen biopsies were cryo-sectioned, samples of around 2000 epithelial or stroma cells microdissected, digested and measured by Orbitrap LC-MS. Proteins were then identified by MS/MS database search and quantified by label-free analysis. An average of 366 protein-groups were identified per sample, and more protein-groups were found in epithelial samples than in stromal samples (442 vs 301, p < 0.0001). Altogether, 1254 distinct protein-groups were found, 289 and 88 of them significantly more often in epithelial and stroma samples, respectively. We assessed five different types of reproducibilities (run-to-run, intrabiopsy, biopsy-to-biopsy, experiment-to-experiment, and patient-to-patient) for protein identification and protein quantification. Reproducibility of protein identification ranged from 78 to 57%, and standard deviation of protein quantification was on patient-to-patient level four times higher than for run-to-run. We conclude that sampling around 2000 cells requires groups of 32 samples to detect significant, over 10-fold differences in protein abundances and thus creates a successful compromise between throughput and quality of results. We therefore believe that this method is suitable for investigating protein-expression profiles during carcinogenesis.
巴雷特食管（BE）与食管腺癌（EAC）的风险增加有关，其特征是正常食管鳞状上皮被柱状上皮取代。这些改变也反映在涉及的细胞类型的蛋白表达谱的变化中。为了分别研究选定细胞类型的蛋白质组，我们结合了激光捕获显微切割（LCM）和液相色谱-质谱（LC-MS）。目的是确定采样方法的灵敏度、特异性和技术重复性，以及活检和患者之间的生物学变异性。冷冻活检被冷冻切片，大约 2000 个上皮或基质细胞的样本进行激光捕获显微切割，消化后用 Orbitrap LC-MS 进行测量。通过 MS/MS 数据库搜索鉴定蛋白质，并通过无标记分析进行定量。每个样本平均鉴定出 366 个蛋白质组，上皮样本中的蛋白质组多于基质样本（442 比 301，p<0.0001）。总共发现了 1254 个不同的蛋白质组，其中 289 个和 88 个分别在上皮和基质样本中更为常见。我们评估了蛋白鉴定和蛋白定量的五种不同的重现性（运行间、活检内、活检间、实验间和患者间）。蛋白鉴定的重现性范围为 78%至 57%，蛋白定量的标准偏差在患者间水平上比运行间水平高四倍。我们得出结论，大约 2000 个细胞的采样需要 32 个样本组来检测蛋白丰度的显著差异（超过 10 倍），从而在通量和结果质量之间取得了成功的平衡。因此，我们认为这种方法适用于研究癌变过程中的蛋白表达谱。
