Proteomic approach to human kidney glomerulus prepared by laser microdissection from frozen biopsy specimens: Exploration of proteome after removal of blood-derived proteins
Proteomic approach to human kidney glomerulus prepared by laser microdissection from frozen biopsy specimens: Exploration of proteome after removal of blood-derived proteins
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DOI：
10.1002/prca.201200016
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发表时间：
2012-08-01
影响因子：
2
通讯作者：
Yamamoto, Tadashi
中科院分区：
文献类型：
--
作者：
Yoshida, Yutaka;Nameta, Masaaki;Yamamoto, Tadashi
目的利用激光显微切割技术从人肾活检标本中提取大量的肾小球血源性蛋白，阻碍了对肾小球蛋白质组的深入研究。我们试图通过从冷冻活检样本中去除血源性蛋白来建立深入分析肾小球蛋白质组的实验平台。实验设计活检标本冰冻切片在激光显微切割前反复PBS洗涤以去除血源性蛋白，并用MS分析肾小球剥离物，通过鉴定蛋白质的动态范围和低丰度蛋白质的检测来评估蛋白质组学分析的深度。结果冰冻切片经两次PBS洗涤后，有效地去除了激光显微切割肾小球中的血源性蛋白质，鉴定出的蛋白质数量增加。用线性离子陷阱-Orbitrap质谱仪对单个标本的肾小球进行分析，得到了400多个识别蛋白质的无冗余、高置信度和高重复性的数据集，从大的动态范围和低丰度蛋白质的识别中可以看出，这些数据集达到了肾小球蛋白质组的相当深度。结论和临床应用PBS清洗冰冻切片成功地去除了血源性蛋白，并对激光显微解剖的肾小球进行了深入的蛋白质组学分析，提示其适用于临床研究。
Purpose Abundance of blood-derived proteins in glomeruli prepared by laser microdissection from human kidney biopsy specimens has hampered in-depth proteomic analysis of glomeruli. We attempted to establish experimental platform for in-depth proteomic analysis of glomeruli by removal of blood-derived proteins from frozen biopsy samples. Experimental design Frozen sections of biopsy samples were exposed to repeated PBS washes prior to laser microdissection to remove blood-derived proteins, and glomerular dissectants were analyzed by MS. The depth of proteomic analysis was evaluated by dynamic range of identified proteins and detection of low-abundance proteins. Results Two times PBS washes of frozen sections effectively eliminated blood-derived proteins in laser-microdissected glomeruli and gave an increased number of identified proteins. Analysis of glomeruli from single specimens by a linear ion trap-Orbitrap mass analyzer generated nonredundant, high-confidence datasets of more than 400 identified proteins with high reproducibility, which attained to a considerable depth of the glomerulus proteome as revealed by a wide dynamic range and identification of low-abundance proteins. Conclusions and clinical relevance Implementation of washing of frozen section with PBS successfully removed blood-derived proteins and resulted in an in-depth proteomic analysis of laser-microdissected glomeruli, suggesting applicability to clinical study.