Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics
Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics
复制标题
DOI：
10.1021/ac049062o
复制
发表时间：
2004-11-15
影响因子：
7.4
通讯作者：
Yoshida, S
中科院分区：
文献类型：
--
作者：
Wada, Y;Tajiri, M;Yoshida, S
在糖蛋白组学中，大量糖蛋白的关键结构问题、蛋白质鉴定、糖基化位点的位置和糖基化位点的微观异质性的评估应该很容易被评估，而质谱学(MS)则提供关于单个纯化的糖蛋白的大量信息。考虑到结构问题是通过研究糖肽来阐明的，并且由几个氨基酸残基组成的胰蛋白酶多肽的串联MS足以用于蛋白质鉴定，因此构建一种基于MS的处理胰酶糖肽的方法将在研究中具有相当大的好处。为此，一种简单有效的方法，即利用碳水化合物基质如纤维素和琼脂糖与低聚糖的亲水结合，成功地应用于胰酶糖肽的分离。通过对基质辅助激光解吸电离(MALDI)产生的离子进行多级串联MS(MSN)分析，确定了多肽和低聚糖的结构如下。由N-连接糖蛋白合成的胰酶糖肽混合物的MALDI离子陷阱质谱图由组成糖肽的[M+H](+)离子组成。糖肽[M+H](+)离子的碰撞诱导解离(CID)产生了糖间隔的峰，间隔例如146、162和203 Da，它们的碎片离子对应于MS2光谱中的肽和肽+N-乙酰氨基葡萄糖(GlcNAc)物种。以糖为间隔的梯形图勾勒出低聚糖的结构，然后将其选作后续MSN分析的前体。对MS2谱中的多肽或多肽+GlcNAc离子或MS1光谱中丰富的相应离子进行CID测定多肽序列，以鉴定蛋白质及其糖基化位点。该方法分离糖肽后进行MSN分析，有效地表征了具有4个N-糖基化位点的β(2)-糖蛋白I的结构，并用于血清总糖蛋白的分析。
In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS (MSn) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the [M + H](+) ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide [M + H](+) ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (GlcNAc) species in the MS2 spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent MSn analyses. The peptide or peptide + GlcNAc ions in the MS2 spectrum or the corresponding ions abundant in the MS1 spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by MSn analysis, efficiently characterized the structures of beta(2)-glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins.