Enhanced analysis of the mouse plasma proteome using cysteine-containing tryptic glycopeptides
Enhanced analysis of the mouse plasma proteome using cysteine-containing tryptic glycopeptides
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DOI：
10.1021/pr0604559
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发表时间：
2007-03-01
影响因子：
4.4
通讯作者：
Simpson, Richard J.
中科院分区：
文献类型：
--
作者：
Bernhard, Oliver K.;Kapp, Eugene A.;Simpson, Richard J.
对小鼠血浆蛋白质组的全面了解对于利用小鼠模型识别人类疾病的蛋白质标记物的研究是重要的。为了加强我们对小鼠血浆蛋白质组的分析，我们开发了一种使用含半胱氨酸的糖肽策略分离低丰度蛋白质的方法。该方法包括两个正交亲和捕获步骤。首先，利用肼化学将糖蛋白偶联到氮杂内酯共聚物凝胶上，然后将半胱氨酸残基进行生物素化。经过胰蛋白酶化和大量洗涤后，用PNGase f从凝胶中释放固定的n -糖基化胰蛋白酶肽，然后用单体亲和素凝胶选择含生物素化半胱氨酸的糖肽，并通过LC-MS/MS进行分析。我们已将该方法应用于小鼠血浆的蛋白质组分析。在C57BL小鼠血浆各200 μ L的两次独立分析中，检测到51种蛋白。当同一血浆样品仅用糖肽分析时，只观察到42种蛋白质。共鉴定出104个n -糖基化位点。其中17个位点迄今尚未在Swiss-Prot数据库中加以注释，而48个位点被认为是可能的、潜在的或相似的，即根据很少或没有实验证据。我们发现，通过含半胱氨酸的糖肽分析，可以检测到低丰度的蛋白质，如表皮生长因子受体、维生素k依赖性蛋白Z、肝细胞生长因子激活剂和淋巴内皮特异性透明质酸受体，因为这些蛋白质在糖肽控制分析中未被检测到。
A comprehensive understanding of the mouse plasma proteome is important for studies using mouse models to identify protein markers of human disease. To enhance our analysis of the mouse plasma proteome, we have developed a method for isolating low-abundance proteins using a cysteine-containing glycopeptide strategy. This method involves two orthogonal affinity capture steps. First, glycoproteins are coupled to an azlactone copolymer gel using hydrazide chemistry and cysteine residues are then biotinylated. After trypsinization and extensive washing, tethered N-glycosylated tryptic peptides are released from the gel using PNGase F. Biotinylated cysteinyl-containing glycopeptides are then affinity selected using a monomeric avidin gel and analyzed by LC-MS/MS. We have applied the method to a proteome analysis of mouse plasma. In two independent analyses using 200 mu L each of C57BL mouse plasma, 51 proteins were detected. Only 42 proteins were seen when the same plasma sample was analyzed by glycopeptides only. A total of 104 N-glycosylation sites were identified. Of these, 17 sites have hitherto not been annotated in the Swiss-Prot database whereas 48 were considered probable, potential, or by similarity - i.e., based on little or no experimental evidence. We show that analysis by cysteine-containing glycopeptides allows detection of low-abundance proteins such as the epidermal growth factor receptor, the Vitamin K-dependent protein Z, the hepatocyte growth factor activator, and the lymphatic endothelium-specific hyaluronan receptor as these proteins were not detected in the glycopeptide control analysis.