Department of Obstetrics and Gynecology, Walter Reed National Military Medical Center, Bethesda, MD, USA.
J Proteome Res. 2011 Nov 4;10(11):5264-71. doi: 10.1021/pr2007736. Epub 2011 Oct 3.
The goal of the present study was to establish a standard operating procedure for mass spectrometry (MS)-based proteomic analysis of laser microdissected (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 ∼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.
本研究的目的是建立一种基于质谱（MS）的激光显微切割（LMD）福尔马林固定、石蜡包埋（FFPE）子宫组织蛋白质组学分析的标准操作程序。对免疫染色乳腺癌 1 型易感性蛋白的大型子宫内膜癌组织微阵列进行高分辨率生物图像分析，能够精确计数细胞，确定子宫内膜癌组织的平均细胞密度为 600 个细胞/nL。我们试图描述通过 LMD 收集的不同体积的组织以及使用本方法进行处理/消化后的肽回收率。我们观察到肽回收率随着组织体积的增加呈近线性增加。基质与恶性上皮之间的肽回收率几乎没有明显差异，且日常回收率没有明显差异。该方法可重复性地从每个 nL 子宫内膜组织中获得 100ng 消化肽，或从每个子宫内膜癌细胞中获得 25pg 肽。使用本方法从 FFPE 组织消化物中评估总肽负荷对柱上总肽和蛋白鉴定数量的影响的液相色谱（LC）-MS/MS 实验结果表明，在所鉴定的肽总数中，总肽负荷的增加在 1000ng 以内呈明显增加趋势，超过该值后，回报递减。此外，我们还观察到，从 LMD 小体积样本获得的等量肽分析中，肽鉴定率不受影响。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方法，用于样品制备，以实现基于活检或手术的组织的 LMD-MS 高通量分析。
