Standardization of a Sample Preparation and Analytical Workflow for Proteomics of Archival Endometrial Cancer Tissue
Standardization of a Sample Preparation and Analytical Workflow for Proteomics of Archival Endometrial Cancer Tissue
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DOI：
10.1021/pr2007736
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发表时间：
2011-11-01
影响因子：
4.4
通讯作者：
Conrads, Thomas P.
中科院分区：
文献类型：
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作者：
Alkhas, Addie;Hood, Brian L.;Conrads, Thomas P.
本研究的目的是建立一个标准的操作程序，用于激光显微切割（LMD）福尔马林固定，石蜡包埋（FFPE）子宫组织的基于质谱（MS）的蛋白质组学分析。对乳腺癌1型易感蛋白免疫染色的大型子宫内膜癌组织微阵列的高分辨率生物图像分析能够精确计数细胞，以确定平均600个细胞/nL的子宫内膜癌组织。我们试图表征通过LMD收集并使用本方法加工/消化的各种体积的组织的肽回收。我们观察到随着解剖的组织体积的增加，肽回收量几乎线性增加。间质上皮与恶性上皮的肽回收率几乎没有明显差异，并且每天的回收率也没有明显差异。该方法可重复地导致每nL子宫内膜组织100 ng消化的肽，或类似于25 pg肽/子宫内膜癌细胞。来自液相色谱（LC）-MS/MS实验以评估柱上的总肽负载对从用本方法制备的FFPE组织电泳物鉴定的肽和蛋白质的总数的影响的结果表明，鉴定的肽的总数明显增加至1000 ng，超过该值观察到收益递减。此外，我们观察到对来自较低体积LMD样品的等效肽量的分析对肽鉴别率没有影响。这些结果表明，这种单管收集-注射蛋白质组学（CTIP）工作流程代表了一种简单、可扩展且高度可靠的样品制备方法，以实现对来自活检或手术的组织的高通量LMD-MS分析。
The goal of the present study was to establish a standard operating procedure for mass spectrometry (MS)-based proteomic analysis of laser micro-dissected (LMD) formalin-fixed, paraffin-embedded (FFPE) uterine tissue. High resolution bioimage analysis of a large endometrial cancer tissue microarray immunostained for the breast cancer type 1 susceptibility protein enabled precise counting of cells to establish that there is an average of 600 cells/nL of endometrial cancer tissue. We sought to characterize the peptide recovery from various volumes of tissue gathered by LMD and processed/digested using the present methodology. We observed a nearly linear increase in peptide recovery amount with increasing tissue volume dissected. There was little discernible difference in the peptide recovery from stromal versus malignant epithelium, and there was no apparent difference in the day-to-day recovery. This methodology reproducibly results in 100 ng of digested peptides per nL of endometrial tissue, or similar to 25 pg peptides/endometrial cancer cell. Results from liquid chromatography (LC)-MS/MS experiments to assess the impact of total peptide load on column on the total number of peptides and proteins identified from FFPE tissue digests prepared with the present methodology indicate a demonstrable increase in the total number of peptides identified up to 1000 ng, beyond which diminishing returns were observed. Furthermore, we observed no impact on the peptide identification rates from analyses of equivalent peptide amounts derived from lower volume LMD samples. These results show that this single-tube collection-to-injection proteomics (CTIP) workflow represents a straightforward, scalable, and highly reliable methodology for sample preparation to enable high throughput LMD-MS analysis of tissues derived from biopsy or surgery.