Review Advances in LC–MS/MS-based glycoproteomics: Getting closer to system-wide site-specific mapping of the N- and O-glycoproteome Morten Thaysen-Andersen ⁎, Nicolle H. Packer Biomolecular Frontiers Research Centre, Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia abstract article info Article history: Received 2 March 2014 Received in revised form 23 April 2014 Accepted 5 May 2014 Available online 12 May 2014 Keywords: Glycoproteomics Glycopeptide Glycoprotein Mass spectrometry Protein glycosylation Glycoproteome Site-specific structural characterization of glycoproteins is important for understanding the exact functional rele- vance of protein glycosylation. Resulting partly from the multiple layers of structural complexity of the attached gly- cans, the system-wide site-specific characterization of protein glycosylation, defined as glycoproteomics, is still far from trivial leaving the N- and O-linked glycoproteomes significantly under-defined. However, recent years have seen significant advances in glycoproteomics driven, in part, by the developments of dedicated workflows and effi- cient sample preparation, including glycopeptide enrichment and prefractionation. In addition, glycoproteomics has benefitted from the continuous performance enhancement and more intelligent use of liquid chromatography and tandem mass spectrometry (LC–MS/MS) instrumentation and a wider selection of specialized software tackling the unique challenges of glycoproteomics data. Together these advances promise more streamlined N- and O-linked glycoproteome analysis. Tangible examples include system-wide glycoproteomics studies detecting thousands of in- tact glycopeptides from hundreds of glycoproteins from diverse biological samples. With a strict focus on the system-wide site-specific analysis of protein N- and O-linked glycosylation, we review the recent advances in LC–MS/MS based glycoproteomics. The review opens with a more general discussion of experimental designs in glycoproteomics and sample preparation prior to LC–MS/MS based data acquisition. Although many challenges still remain, it becomes clear that glycoproteomics, one of the last frontiers in proteomics, is gradually maturing en- abling a wider spectrum of researchers to access this new emerging research discipline. The next milestone in ana- lytical glycobiology is being reached allowing the glycoscientist to address the functional importance of protein glycosylation in a system-wide yet protein-specific manner. © 2014 Elsevier B.V. All rights reserved. 1. Introduction to system-wide site-specific analysis of protein glycosylation 1.1. Structure and function of protein N- and O-glycosylation Protein glycosylation is the covalent attachment of complex carbohy- drates or oligosaccharides (here collectively called glycans) to specific amino acid residues of the polypeptide backbone of proteins. The biosyn- thetic machinery of mammals primarily allows the attachment of glycans to asparagine (N-glycosylation) and serine/threonine (O-glycosylation) residues thereby forming two major classes of protein glycosylation. These types i.e. N-GlcNAc and O-GalNAc (mucin-type) glycosylation, which are both the focus of this review, are synthesized in a non- template driven manner by a spectrum of glycosylation enzymes through different routes in the secretory pathway. The N-linked glycans, which in mammals are usually restricted to occupy asparagine res- idues in NXS/T, X ≠ P consensus sequences (sequons), consist of a common chitobiose core (Man 3 GlcNAc 2 ) from which a variety of monosaccharides and other glycan modifications may be added to the non-reducing termini in speci fic linkage configurations [1]. The status of the cellular glycosylation machinery and the nature of the proteins undergoing glycosylation together determine the repertoire of glycans being presented on the protein carriers thus creating the important features of cell- and protein-specific glyco- sylation [2]. N-linked glycans are usually larger by mass and volume than their O-linked counterparts, which, in contrast, are more Biochimica et Biophysica Acta 1844 (2014) 1437–1452 Abbreviations: CID, collision induced dissociation; Con A, concanavalin A; CSF, cerebrospinal fluid; DDA, data dependent acquisition; DIA, data independent acquisition; ECD, electron capture dissociation; EIC (or XIC), extracted ion chromatogram; ESI, electrospray ionization; ETD, electron transfer dissociation; FDR, false discovery rate; FT-ICR, Fourier transform ion cyclotron reso- nance; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; HCD, higher-energy collisional dissociation; HILIC, hydrophilic interaction liquid chromatog- raphy; HPLC, high performance liquid chromatography; IRMPD, infrared multiphoton dissociation; LC–MS/MS, liquid chromatography tandem mass spectrometry; LTQ, linear trap quadrupole; MALDI, matrix assisted laser desorption ionization; Man, mannose; MRM, multiple reaction monitoring; MS n , mass spectrometry to the nth power; nETD, negative electron transfer dissociation; NeuAc, N-acetyl-5-neuraminic acid; Q-TOF, quadrupole-time-of-flight; RP, reversed phase; SPE, solid phase extraction; TMT, tandem mass tag; UPLC, ultra-high pressure liquid chromatography ⁎ Corresponding author at: Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia. Tel.: +61 2 9850 7487; fax: +61 2 9850 6192. E-mail address: morten.andersen@mq.edu.au (M. Thaysen-Andersen). http://dx.doi.org/10.1016/j.bbapap.2014.05.002 1570-9639/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap