Deep amino acid sequencing of native brain GABAA receptors using high-resolution mass spectrometry.
Deep amino acid sequencing of native brain GABAA receptors using high-resolution mass spectrometry.
复制标题
DOI：
10.1074/mcp.m111.011445
复制
发表时间：
2012-01
期刊：
影响因子：
--
通讯作者：
Evers AS
中科院分区：
文献类型：
--
作者：
Chen ZW;Fuchs K;Sieghart W;Townsend RR;Evers AS
低丰度、完整的膜蛋白，特别是跨膜结构域的质谱测序提出了跨越样品制备的多个阶段的挑战，包括溶解、纯化、酶消化、肽提取和色谱分离。我们描述了一种方法，通过该方法，我们从2皮摩尔的亲和纯化的大鼠大脑新皮层GABAA受体中获得了12个γ-氨基丁酸A型受体（GABAA受体）亚基的高肽覆盖率。针对α1亚基，我们使用数据库搜索算法从片段化光谱（MS 2）中鉴定了覆盖96%蛋白质序列的肽，并从Orbitrap光谱的从头测序中推断出蛋白质中80%的氨基酸残基。该工作流程结合了微尺度膜蛋白溶解、蛋白脱脂、溶液中多酶消化、用于肽提取的多个固定相以及高分辨率全扫描和碎片化光谱的采集。对于含有跨膜结构域的肽的从头测序，使用胰凝乳蛋白酶的定时双酶切来产生具有重叠序列的肽，然后通过使用C4随后使用多孔石墨碳固定相的顺序固相萃取来回收所述肽。肽鉴定和氨基酸残基序列的特异性通过高质量准确度和对母体和碎片离子的电荷状态分配而增加。对三个单独的脑样本的分析表明，在所有三个重复中观察到78%的α1亚基序列，在三个重复中的两个重复中覆盖了另外13%，表明高度的序列覆盖重现性。采用无标记定量分析法测定11种γ-氨基丁酸A型受体亚基的相对丰度。深度序列MS数据还揭示了α1亚基上的两个N-糖基化位点，证实了γ2亚基的两个剪接变体（γ2L和γ2S），并解决了α5亚基序列中的数据库差异。
Mass spectrometric sequencing of low abundance, integral membrane proteins, particularly the transmembrane domains, presents challenges that span the multiple phases of sample preparation including solubilization, purification, enzymatic digestion, peptide extraction, and chromatographic separation. We describe a method through which we have obtained high peptide coverage for 12 γ-aminobutyric acid type A receptor (GABAA receptor) subunits from 2 picomoles of affinity-purified GABAA receptors from rat brain neocortex. Focusing on the α1 subunit, we identified peptides covering 96% of the protein sequence from fragmentation spectra (MS2) using a database searching algorithm and deduced 80% of the amino acid residues in the protein from de novo sequencing of Orbitrap spectra. The workflow combined microscale membrane protein solubilization, protein delipidation, in-solution multi-enzyme digestion, multiple stationary phases for peptide extraction, and acquisition of high-resolution full scan and fragmentation spectra. For de novo sequencing of peptides containing the transmembrane domains, timed digestions with chymotrypsin were utilized to generate peptides with overlapping sequences that were then recovered by sequential solid phase extraction using a C4 followed by a porous graphitic carbon stationary phase. The specificity of peptide identifications and amino acid residue sequences was increased by high mass accuracy and charge state assignment to parent and fragment ions. Analysis of three separate brain samples demonstrated that 78% of the sequence of the α1 subunit was observed in all three replicates with an additional 13% covered in two of the three replicates, indicating a high degree of sequence coverage reproducibility. Label-free quantitative analysis was applied to the three replicates to determine the relative abundances of 11 γ-aminobutyric acid type A receptor subunits. The deep sequence MS data also revealed two N-glycosylation sites on the α1 subunit, confirmed two splice variants of the γ2 subunit (γ2L and γ2S) and resolved a database discrepancy in the sequence of the α5 subunit.