Systems Approaches to Health and Disease 1307 Glycoproteomics: a powerful tool for characterizing the diverse glycoforms of bacterial pilins and flagellins Paul G. Hitchen*†, Katie Twigger*, Esmeralda Valiente‡, Rebecca H. Langdon‡, Brendan W. Wren†‡ and Anne Dell*† 1 *Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, U.K., †Centre for Integrative Systems Biology, Imperial College, London SW7 2AZ, U.K., and ‡Department of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. Abstract With glycosylation now firmly established across both Archaeal and bacterial proteins, a wide array of glycan diversity has become evident from structural analysis and genomic data. These discoveries have been built in part on the development and application of mass spectrometric technologies to the bacterial glycoproteome. This review highlights recent findings using high sensitivity MS of the large variation of glycans that have been reported on flagellin and pilin proteins of bacteria, using both ‘top down’ and ‘bottom up’ approaches to the characterization of these glycoproteins. We summarize current knowledge of the sugar modifications that have been observed on flagellins and pilins, in terms of both the diverse repertoire of monosaccharides observed, and the assemblage of moieties that decorate many of these sugars. Introduction Glycosylation, the post-translational modification of pro- teins by carbohydrates, has long been recognised as a fundamental strategy used by eukaryotes to influence and modulate protein structure and function [1]. It is now evident that protein glycosylation is abundant in prokaryotes, with an ever increasing number of glycoproteins being identified. Sugars present on glycoproteins have traditionally been studied once released from the protein, but new technologies are now permitting the analysis of sugars in situ using MS [2]. The earliest examples of protein glycosylation in prokaryotes were found in the Archaea, which express glycosylated surface (S-layer) proteins [3], and a number of S-layer glycans have now been reported [4]. Subsequently, protein glycosylation was identified in Bacteria such as Flavobacterium meningosepticum [5,6] and Neisseria meningitidis [6,7], but was largely ignored as a rare curiosity until the discovery of a general N-glycosylation system in Campylobacter jejuni following the completion of its genome sequence [8]. It is now firmly established that glycosylation is widespread across both Archaea and bacteria [9]. Indeed, with the progress in sequencing of bacterial genomes, it has become more evident that shared genes among pathogenic bacteria are involved in the glycosylation process and that there is a commonality of sugars across bacteria. An example of commonality is the O-glycosylation of Neisseria pilin and the Key words: bacterium, flagellin, glycoproteomics, glycosylation, mass spectrometry, pilin. Abbreviations used: CAD-MS/MS, collisionally assisted dissociation tandem MS; DATDH, 2,4-diacetamido-2,4,6-trideoxyhexose, ES-MS, electrospray MS; ETD-MS/MS, electron transfer dissociation tandem MS; HexNAc, N-acetylhexosamine; LPS, lipopolysaccharide; O-GlcNAc, β-O- linked N-acetylglucosamine. 1 To whom correspondence should be addressed (email a.dell@imperial.ac.uk). N-glycosylation of Campylobacter flagellin, where the sugar DATDH (2,4-diacetamido-2,4,6-trideoxyhexose) links the glycan to the protein in both cases [10]. These two pathogens share glycosylation genes (pgl) involved in the synthesis and transfer of DATDH onto the protein backbone [11]. The MS glycosylation studies in Campylobacter recognised that ES-MS (electrospray MS) analysis of glycopeptides yielded abundant signature fragment ions for the nitrogen-containing sugars, and this has facilitated their discovery in other organ- isms. For example, work in our laboratory has characterized the sugar DATDH in Wolinella succinogenes (P.G. Hitchen and A. Dell), which was predicted from the presence of pgl genes in the genome sequence [12]. MS analysis has revealed the presence of a hexasaccharide attached to the protein via the DATDH residue, which was first noted from the observation of the characteristic signature oxonium ion (m/z 229) for DATDH. To date, the study of prokaryotic protein glycosylation in bacteria has been best exemplified by the flagellin and pilin glycoproteins [13,14]. Flagellins and pilins are proteins that are arranged into large filamentous structures that extrude from the bacterial surface and are termed the flagellum and pilus respectively. The flagellum is important for bacterial virulence, motility and colonization. Short pilus, termed fimbriae, are used to attach the pathogen to the host surface, whereas type IV pili are integral for adherence and motility. Although the exact function of glycosylation of these appendages has yet to be fully understood, it is evident that glycosylation is functionally important. For example, mutants in glycosylation of Campylobacter flagellin are non-motile and accumulate flagellin intracellularly and have also been shown to display limited flagellin glycoforms that Biochem. Soc. Trans. (2010) 38, 1307–1313; doi:10.1042/BST0381307 C  The Authors Journal compilation C  2010 Biochemical Society