Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

B American Society for Mass Spectrometry, 2017 J. Am. Soc. Mass Spectrom. (2017) 28:1751Y1764 DOI: 10.1007/s13361-017-1701-4 FOCUS: USING ELECTRONS AND RADICAL CHEMISTRY TO CHARACTERIZE BIOLOGICAL MOLECULES: RESEARCH ARTICLE Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization Qing Yu,1 Bowen Wang,2 Zhengwei Chen,3 Go Urabe,2 Matthew S. Glover,1,4 Xudong Shi,2 Lian-Wang Guo,2 K. Craig Kent,5 Lingjun Li1,3,4 1 School of Pharmacy, University of Wisconsin, Madison, WI 53705, USA Department of Surgery, Wisconsin Institutes for Medical Research, Madison, WI 53705, USA 3 Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA 4 Cardiovascular Research Center Training Program in Translational Cardiovascular Science, University of Wisconsin-Madison, Madison, WI 53705, USA 5 The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA 2 Abstract. Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum, and that can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides, which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study, a redefined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding five glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid arteries over the course of restenosis progression to investigate the potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to largescale O-glycoproteome characterization. Keywords: Glycopeptide, Electron-transfer dissociation, EThCD, High-energy collision dissociation, Glycoproteomics, Glycosylation Received: 30 December 2016/Revised: 28 March 2017/Accepted: 29 April 2017/Published Online: 10 July 2017 Introduction P rotein glycosylation is the covalent attachment of complex carbohydrates or oligosaccharides, collectively called gly- Electronic supplementary material The online version of this article (doi:10. 1007/s13361-017-1701-4) contains supplementary material, which is available to authorized users. Correspondence to: Lingjun Li; e-mail: cans, to specific amino acid residues of the polypeptide backbone of proteins. Being one of the most common posttranslational modifications (PTMs), glycosylation is estimated to occur in more than half of all eukaryotic proteins [1], with two major types being N-glycosylation and O-glycosylation [2]. It is involved in biological processes such as cell adhesion, signaling, inflammatory response, as well as a wide variety of pathologic states [2–4]. The ability to accurately characterize the structure of glycoproteins is of great importance to unravel the diverse functions 1752 of glycosylation, especially for N-glycosylation that can be highly heterogeneous. However, N-glycosylated proteins are most often highly complex by presenting a multitude of diverse sugar–amino acid combinations and characterization with MS remains a great analytical challenge due to this heterogeneity [4, 5]. Structural characterization encompasses N-glycoprotein/ glycopeptide identification, locations of attachment sites, and evaluation of glycosylation site micro-heterogeneity. Most of the previous N-glycoproteomic studies involved enzymatic removal of glycans, and separate identifications of glycans and amino acid sequence [6–10]. Although there are multiple well-established protocols, detaching glycans from their peptide backbones accompanies loss of multiple layers of information (i.e., amino acid site-specific glycoform and microheterogeneity) [11, 12]. The ideal way to comprehensively characterize glycopeptides, especially in a large-scale glycoproteomics context, is to preserve the glycan side chain on the peptide and collect glycan and peptide fragmentation information simultaneously [13, 14]. However, due to its heterogeneous nature, fragmenting an intact glycopeptide has always been difficult and yet no single fragmentation technique is able to generate a complete picture in a single MS/MS spectrum [6, 7, 14, 15]. Peaks resulting from glycosidic bond cleavages dominate spectra generated by collision-induced dissociation (CID) with little knowledge of glycosylation sites and amino acid sequences, whereas c/z-ion series in ETD type of experiments yield the glycosylation site and peptide identity with little information on glycan side chain composition [16, 17]. Higher-energy collision dissociation produces abundant diagnostic oxonium ions and partial glycopeptide information as the collision energy is more evenly distributed, enabling intact glycopeptide identification in some cases, most of which are studies on purified target proteins [16–18]. Although there have been multiple reports showing the capabilities of using CID or ETD alone to sequence sugar-modified peptides, most often the data were collected only with a few protein standards that were modified by less complex glycation or O-glycosylation. It is not applicable to large-scale characterization of much more branched and complex glycosylated peptides, such as N-glycopeptides where glycan fragments dominate MS/MS spectra, and it is especially challenging to work with complex proteome samples [19–24]. An alternative approach for a more detailed characterization of glycopeptides combines MS/MS and MS3 experiments with CID or HCD. In this approach, the glycopeptide ion is selected and fragmented, resulting in a variety of fragment ions predominantly attributable to the cleavage of glycosidic linkages. The peptide ion carrying a single HexNAc, is subjected to a second ion isolation/fragmentation cycle, resulting in fragmentation of the peptide moiety [25–28]. However, this also requires prior knowledge of the targeted peptides to select for MS3, thus limiting its throughput [25–28]. Several other reports alternate collision energies and collect sequential MS/MS on the same precursor, Q. Yu et al.: EThcD-Enabled Intact Glycopeptide Analys (...truncated)