Site-specific N-glycosylation analysis:: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides
Site-specific N-glycosylation analysis:: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides
复制标题
DOI：
10.1002/rcm.1585
复制
发表时间：
2004-01-01
影响因子：
2
通讯作者：
Perreault, H
中科院分区：
文献类型：
--
作者：
Krokhin, O;Ens, W;Perreault, H
通常可以通过蛋白水解消化、分离、质谱 (MS) 和串联 MS (MS/MS) 的组合来鉴定蛋白质中的糖基化位点。液相色谱 (LC) 与 MS/MS 相结合已成为检测消化混合物中糖肽以及指定糖基化位点和糖肽序列的可靠方法。 LC 与 MS 的直接连接依赖于电喷雾电离，对于大多数蛋白水解肽和糖肽来说，电喷雾电离会产生带有两个、三个或四个电荷的离子。如果不使用单电荷水平的解卷积，此类糖肽离子的 MS/MS 谱通常会导致不明确的解释。相比之下，基质辅助激光解吸/电离 (MALDI) 技术通常产生单电荷肽和糖肽离子。这些离子需要扩展的 m/z 范围，如这些实验中使用的四极杆-四极杆飞行时间 (QqTOF) 仪器提供的那样，但研究单电荷离子的主要优点是 MS/MS 谱图的简单性和一致性。本研究的首要目标是开发识别和使用糖肽 [M+H](+) 离子作为 MS/MS 前体的方法，从而将糖肽/糖蛋白鉴定作为更广泛的蛋白质组学研究的一部分。其次，本文旨在证明 N-糖肽 MALDI-MS/MS 谱的实用性。制备商业获得的不同类型蛋白质的混合物并进行还原、烷基化和胰蛋白酶消化。微柱反相分离允许在 MALDI 板上沉积多个组分，然后对所有肽进行 MS 和 MS/MS 分析。 MS 后通过糖型之间特定的 m/z 间距模式（162、203、291 u）鉴定糖肽组分，然后通过 MS/MS 进行分析。在大多数情况下，糖肽 [M+H](+) 离子的 MS/MS 谱图具有可用于确定糖组成、肽序列以及可能的糖基化位点的峰。肽相关产物离子可用于数据库搜索程序并允许识别糖蛋白。版权所有 (C) 2004 John Wiley Sons, Ltd.
The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides. MS/MS spectra of such glycopeptide ions often lead to ambiguous interpretation if deconvolution to the singly charged level is not used. In contrast, the matrix-assisted laser desorption/ionization (MALDI) technique usually produces singly charged peptide and glycopeptide ions. These ions require an extended m/z range, as provided by the quadrupole-quadrupole time-of-flight (QqTOF) instrument used in these experiments, but the main advantages of studying singly charged ions are the simplicity and consistency of the MS/MS spectra. A first aim of the present study is to develop methods to recognize and use glycopeptide [M+H](+) ions as precursors for MS/MS, and thus for glycopeptide/glycoprotein identification as part of wider proteomics studies. Secondly, this article aims at demonstrating the usefulness of MALDI-MS/MS spectra of N-glycopeptides. Mixtures of diverse types of proteins, obtained commercially, were prepared and subjected to reduction, alkylation and tryptic digestion. Micro-column reversed-phase separation allowed deposition of several fractions on MALDI plates, followed by MS and MS/MS analysis of all peptides. Glycopeptide fractions were identified after MS by their specific m/z spacing patterns (162, 203, 291 u) between glycoforms, and then analyzed by MS/MS. In most cases, MS/MS spectra of [M+H](+) ions of glycopeptides featured peaks useful for determining sugar composition, peptide sequence, and thus probable glycosylation site. Peptide-related product ions could be used in database search procedures and allowed the identification of the glycoproteins. Copyright (C) 2004 John Wiley Sons, Ltd.