基于凝胶的重组γ-氨基丁酸A（GABA(A)）受体亚基的质谱分析，该受体亚基为强疏水性跨膜蛋白。

Gel-based mass spectrometric analysis of recombinant GABA(A) receptor subunits representing strongly hydrophobic transmembrane proteins.

作者信息

Kang Sung Ung, Fuchs Karoline, Sieghart Werner, Lubec Gert

机构信息

Department of Pediatrics, Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Austria.

出版信息

J Proteome Res. 2008 Aug;7(8):3498-506. doi: 10.1021/pr800236u. Epub 2008 Jun 19.

DOI:10.1021/pr800236u

PMID:18563923

Abstract

GABA(A) receptors are the major inhibitory transmitter receptors in mammalian brain and are composed of several protein subunits that can belong to different subunit classes, leading to enormous heterogeneity. To establish techniques for the analysis of GABA(A) receptors in complex mixtures such as brain tissue, recombinant receptors composed of alpha1 and His-tagged beta3 subunits expressed in insect cells were purified by affinity chromatography and run on blue native gels. After denaturing, receptors were subjected to one- or two-dimensional electrophoresis in SDS-gels. Proteins were cleaved by multienzyme proteolysis and subjected to nano-ESI-LC-MS/MS. Both GABA(A) receptor subunits were well-separated and unambiguously identified by sequence coverage of 99.1% (alpha1) and 92.9% (beta3).

摘要

GABA(A)受体是哺乳动物大脑中的主要抑制性递质受体，由几个可属于不同亚基类别的蛋白质亚基组成，导致极大的异质性。为建立分析复杂混合物（如脑组织）中GABA(A)受体的技术，通过亲和色谱法纯化了在昆虫细胞中表达的由α1和His标签β3亚基组成的重组受体，并在蓝色天然凝胶上进行电泳。变性后，受体在SDS凝胶中进行一维或二维电泳。蛋白质通过多酶蛋白水解进行切割，并进行纳米电喷雾液相色谱-串联质谱分析。两个GABA(A)受体亚基均得到很好的分离，并通过99.1%（α1）和92.9%（β3）的序列覆盖率得到明确鉴定。