A method for proteomic identification of membrane-bound proteins containing Asn-linked oligosaccharides
A method for proteomic identification of membrane-bound proteins containing Asn-linked oligosaccharides
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DOI：
10.1016/j.ab.2004.05.038
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发表时间：
2004-09-01
影响因子：
2.9
通讯作者：
Mahuran, DJ
中科院分区：
文献类型：
--
作者：
Fan, XL;She, YM;Mahuran, DJ
细胞表面的糖基化蛋白已被证明是细胞间相互作用在发育和分化中必不可少的。我们的最终目标是鉴定直接参与这些关键体内功能的天冬酰胺连接的寡糖。因为这样的寡糖将被期望驻留在完整的质膜蛋白，和传统的二维凝胶技术是无效的，在分离这样的蛋白质，我们已经开发了一种新的方法，他们的识别上的蛋白质组学规模从秀丽隐杆线虫。将膜蛋白溶解在盐酸胍中，沉淀并用胰蛋白酶消化。然后通过凝集素色谱法分离糖肽。接下来，糖肽酶F消化从肽中除去寡糖，并将连接的每个Asn转化为Asp。然后通过基质辅助激光解吸/电离四极杆飞行时间（MALDI-Q-TOF）质谱法分析肽。因此，通过这些肽的序列标签和在Asn-X-Ser/Thr共有序列处至少一个推断的Asn残基转化为Asp来鉴定膜糖蛋白。为了验证这种方法的实用性，我们已经确定了13个膜结合的N-糖基化的蛋白质从我们的总糖肽馏分的MALDI-Q-TOF分析上观察到的主峰。(C)2004年爱思唯尔公司All rights reserved.
Glycosylated proteins on the cell surface have been shown to be essential for cell-cell interactions in development and differentiation. Our ultimate goal is to identify Asn-linked oligosaccharides that are directly involved in these critical in vivo functions. Because such oligosaccharides would be expected to reside on the integral plasma membrane proteins, and conventional two-dimensional gel techniques are ineffective at separating such proteins, we have developed a new approach to their identification on a proteomics scale from Caenorhabditis elegans. Membrane proteins are solubilized in guanidine-HCl, precipitated, and digested with trypsin. The glycopeptides are then separated by lectin chromatography. Next, glycopeptidase F digestion removes the oligosaccharides from the peptides and converts to Asp each Asn to which one was attached. The peptides are then analyzed by matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) mass spectrometry. Thus, the membrane glycoproteins are identified through the sequence tags of these peptides and the conversion of at least one deduced Asn residue to Asp at the Asn-X-Ser/Thr consensus sequence. To validate the utility of this approach, we have identified 13 membrane-bound N-glycosylated proteins from the major peaks observed on MALDI-Q-TOF analysis of our total glycopeptide fraction. (C) 2004 Elsevier Inc. All rights reserved.