Characterization of glomerular extracellular matrix by proteomic analysis of laser-captured microdissected glomeruli
Characterization of glomerular extracellular matrix by proteomic analysis of laser-captured microdissected glomeruli
复制标题
DOI：
10.1016/j.kint.2016.09.044
复制
发表时间：
2017-02-01
影响因子：
19.6
通讯作者：
Merchant, Michael L.
中科院分区：
文献类型：
--
作者：
Hobeika, Liliane;Barati, Michelle T.;Merchant, Michael L.
细胞外基质（ECM）异常重塑是许多肾小球疾病的显著特征，是肾小球损伤的最终共同途径。然而，ECM组成的变化伴随着疾病相关的重塑是未知的。ECM的物理性质为使用标准蛋白质提取技术表征组合物带来了挑战，因为ECM的不溶性组分经常被丢弃，并且许多ECM蛋白质与其他细胞蛋白质相比丰度低。先前定义正常ECM组成的蛋白质组学研究使用了大量从用于移植或通过肾切除术用于癌症的人肾中分离的肾小球。在这里，我们检查的能力，以确定ECM蛋白质的质谱使用肾小球部分兼容的标准肾活检标本。通过与Matrisome数据库和先前鉴定的肾小球ECM蛋白进行比较，将蛋白质分类为ECM。最佳的ECM蛋白鉴定导致连续脱细胞和蛋白质提取的100人肾小球切片分离的激光捕获显微切割冷冻或福尔马林固定，石蜡包埋的组织。总共鉴定了147种ECM蛋白，包括先前鉴定的大多数结构和GBM蛋白以及先前不与肾小球相关的许多基质和肾小球基底膜蛋白。因此，我们的研究证明了从肾活检组织分离的肾小球切片中提取的肾小球ECM蛋白质组学分析的可行性，并扩展了肾小球中已知ECM蛋白的列表。
Abnormal extracellular matrix (ECM) remodeling is a prominent feature of many glomerular diseases and is a final common pathway of glomerular injury. However, changes in ECM composition accompanying disease-related remodeling are unknown. The physical properties of ECM create challenges for characterization of composition using standard protein extraction techniques, as the insoluble components of ECM are frequently discarded and many ECM proteins are in low abundance compared to other cell proteins. Prior proteomic studies defining normal ECM composition used a large number of glomeruli isolated from human kidneys retrieved for transplantation or by nephrectomy for cancer. Here we examined the ability to identify ECM proteins by mass spectrometry using glomerular sections compatible with those available from standard renal biopsy specimens. Proteins were classified as ECM by comparison to the Matrisome database and previously identified glomerular ECM proteins. Optimal ECM protein identification resulted from sequential decellularization and protein extraction of 100 human glomerular sections isolated by laser capture microdissection from either frozen or formalin-fixed, paraffin-embedded tissue. In total, 147 ECM proteins were identified, including the majority of structural and GBM proteins previously identified along with a number of matrix and glomerular basement membrane proteins not previously associated with glomeruli. Thus, our study demonstrates the feasibility of proteomic analysis of glomerular ECM from retrieved glomerular sections isolated from renal biopsy tissue and expands the list of known ECM proteins in glomeruli.