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REVIEW ARTICLE Mucin-type O-glycosylation – putting the pieces together Pia H. Jensen, Daniel Kolarich and Nicolle H. Packer Department of Chemistry and Biomolecular Sciences, Faculty of Science, Biomolecular Frontiers Research Centre, Macquarie University, Sydney, Australia Introduction Protein glycosylation is known to be involved in cellu- lar targeting and secretion [1]. It can also help to regu- late enzymatic activity, confer enhanced stability and solubility to secreted proteins, and affect the function- ality of proteins in the immune system. Moreover, gly- coproteins participate in cell–cell and cell–matrix interactions, and mediate complex developmental func- tions [2]. Glycosylation is one of the major types of post-translational modification that proteins can undergo. In fact, 13 different monosaccharides and eight amino acids have been reported across species to be involved in glycoprotein linkages [3]. The two major types of oligosaccharide attachment to the protein are referred to as N-linked and O-linked glycosylation. N-linked oligosaccharides are usually attached via a GlcNAc linkage to Asn in the consensus sequence NXT ⁄ S (C) (X „ P). O-linked oligosaccharides, how- ever, can be variously attached to Ser or Thr via O-linkages to fucose, Glc, mannose, xylose and other sugars, as well as to the most commonly found mucin- type O-linked a-GalNAc. Note that the single O-linked b-GlcNAc attached to the hydroxyl group of Ser and ⁄ or Thr, and has been found to be a cytoplasmic signalling modification, similar to phosphorylation Keywords electron transfer dissociation (ETD) ⁄ electron capture dissociation (ECD); glycopeptides; MS; mucin oligosaccharides; O-glycosylation; released glycans; site specificity Correspondence N. Packer, Department of Chemistry and Biomolecular Sciences, Faculty of Science, Biomolecular Frontiers Research Centre, Macquarie University, Building E8C, Room 307, Sydney, NSW, 2109, Australia Fax: +61 2 9850 8313 Tel: +61 2 98508176 E-mail: nicki.packer@mq.edu.au Website: http://www.chem.mq.edu.au/ academics/npacker.html (Received 12 June 2009, revised 3 September 2009, accepted 11 September 2009) doi:10.1111/j.1742-4658.2009.07429.x The O-glycosylation of Ser and Thr by N-acetylgalactosamine-linked (mucin-type) oligosaccharides is often overlooked in protein analysis. Three characteristics make O-linked glycosylation more difficult to analyse than N-linked glycosylation, namely: (a) no amino acid consensus sequence is known; (b) there is no universal enzyme for the release of O-glycans from the protein backbone; and (c) the density and number of occupied sites may be very high. For significant biological conclusions to be drawn, the complete picture of O-linked glycosylation on a protein needs to be deter- mined. This review specifically addresses the analytical approaches that have been used, and the challenges remaining, in the characterization of both the composition and structure of mucin-type O-glycans, and the determination of the occupancy and heterogeneity at each amino acid attachment site. Abbreviations CID, collision-induced dissociation; CR, charge-reduced; ECD, electron capture dissociation; ETD, electron transfer dissociation; GalNAc, N-acetylgalactosamine; HexNAc, N-acetlyhexosamine; O-GlcNAc, O-linked GlcNAc. FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS 81 [4,5]. We mention this linkage here because it may be mistaken, by scientists new to the field, as a mucin- type glycosylation, because of its equivalent mass [N-acetlyhexosamine (HexNAc)]. The transfer of GalNAc from UDP-GalNAc to Ser or Thr is catalysed by polypeptide N-acetyl-a-d-galac- tosaminyltransferases [6–8]. These enzymes are sequen- tially and functionally conserved across species [9,10], as well as being differentially expressed over tissue and time, suggesting complex and strict regulation. There are up to 20 different known isoforms of polypeptide N-acetyl-a-d-galactosaminyltransferases. They are dif- ferentially expressed, and many have clear specificities for the sites of attachment of the GalNAc to Ser ⁄ Thr. This diversity determines the density and site occu- pancy of the mucin-type O-glycosylation [11,12]. Attachment of the initial GalNAc occurs in the Golgi, to the completely folded protein, and this starts the action of numerous glycosyltransferases that result in the extension of the GalNAc into numerous different O-glycan structures. The enzymes responsible for this diversification of the O-glycans are very specific in their activity, and their functional importance has been reviewed [13,14]; however, it is beyond the scope of this minireview to discuss them in detail. O-glycans are known to be associated with many known, and many yet to be defined, critical biological functions. Alteration of mucin-type O-glycosylation pathways in animal models leads to diverse effects, ranging from embryonic death to developmental defects and disease. Mutations or other factors that specifically change or inhibit O-linked glycosylation of proteins have been associated with a variety of differ- ent diseases, such as familial tumoral calcinosis (hyper- phosphataemia leading to the development of calcified masses in soft tissues) [15,16], Tn syndrome (haemoly- sis of a subset of haematopoietic cells, leading to thrombocytopenia and haemolytic anaemia) [17,18], IgA nephropathy [19–21], high-density lipoprotein metabolism [22,23], and tumour formation and meta- stasis [24–26]. Additionally, it has been associated with altered immune response, mostly due to altered adhesive properties resulting in decreased rolling on P-selectins, E-selectins, and L-selectins [27]. Changes in O-glycosylation specifically on the high molecular weight mucin glycoproteins have been impli- cated in processes as varied as inflammatory responses, angiogenesis, autoimmunity, and cancer. The mucins are highly O-glycosylated proteins found in secretions and mucous membranes and characterized by repeat sequence domains that have a high frequency of Ser and Thr residues carrying a large number of glycans in very close proximity [28]. The mucins and their glyco- sylation have been implicated in many types of cancers (e.g. aberrant glycosylation of MUC1 in breast cancer [29]), and are the targets of recognition by many tumour-specific antibodies against glycans. The biolog- ical significance of mucin-type O-glycosylation is, how- ever, outside the scope of this review, and the interested reader would be well advised to consult the recent review by Tian and Ten Hagen [14]. This minireview will focus expressly on the analytical technologies currently available for analysis of the major mammalian types of mucin-type O-linked GalNAc- linked glycosylation. The content is designed to give newcomers to this field an introduction to what can be done, and what is still challenging, in the analysis of these specific, heterogeneous protein modifications. What makes O-glycan analysis challenging? We believe that there are a variety of reasons why O-linked protein glycosylation has been overlooked in analysis as compared with N-linked glycans, as follows. First, mucin-type O-glycosylation lacks a known amino acid consensus sequence. In contrast to N-gly- cosylated sites, O-glycosylated sites do not reside in a known amino acid sequence. Several prediction tools have been developed and improved over time [30–33], but none of them is very satisfying. It appears that the lack of validated site glycosylation data is the biggest barrier to developing a useful predictor. Second, there is no enzyme for universal O-glycan release from the protein. System-wide analysis of mucin-type O-glycosylation remains a challenge, owing to structural heterogeneity and the lack of specific enzymatic tools comparable to N-glycosidase F or N-glycosidase A. A general endo-N-acetylgalactosaminy l- transferase activity has been reported [34], but the commercially available O-glycanase has a specificity restricted to the disaccharide sequence Gal–GalNAc only [35], and therefore resistance to O-glycanase should not be taken as evidence for the lack of O-linked saccharide chains. The third reason concerns glycan heterogeneity on glycosylation sites. Mucin-type O-glycosylation is very heterogeneous, and there is no general detection or isolation method to accommodate this [36]. Several attempts to metabolically incorporate tags on the glycans have been successful [37,38], but have been limited to cell culture and animal studies. Note that it has been shown that the O-glycosylation pattern of insect cell lines changes with alterations in culture media [39]. Mucin-type O-glycosylation P. H. Jensen et al. 82 FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS The different glycoproteomic approaches to the characterization of glycoproteins were recently described by Dodds et al. [40]. They divided the field into three major parts: (a) the proteocentric branch, which uses glycosylation as a means of enriching a subset of glycoproteins, only to cleave off the glycan in order to identify the proteins; (b) the glycocentric branch, which looks only at the glycans released from a protein or subset of proteins; and (c) the reductionist glycoproteomics branch, which analyses both protein and attached glycans, but is limited to studying one or a few proteins. The authors stress the need to develop real global glycoproteomic analysis tools to character- ize both N-glycosylation and O-glycosylation on all proteins of interest. This review attempts to give an overview of the methods currently used in what is arguably the last frontier of glycoanalysis – mucin-type O-glycosylation. Screening of intact O-glycoproteins – what we can do Lectins and antibodies are often used for screening and comparing the glycosylation of large sample sets of intact proteins. This may be performed either by histology of tissue samples [41] or on arrays of extracted proteins [42–44]. These types of analyses are high-throughput as well as fairly reproducible, which is useful when multiple proteins in multiple samples are being compared [44]. They provide a broad profil- ing that monitors changes in many glycans on many proteins. It is important to keep in mind, however, that little structural data can be obtained from lectin studies alone [45]. Jacalin is generally regarded as an O-glycan specific lectin, but has been shown to bind N-glycosylated proteins as well [46]. Additionally, the specificity of lectins can be complicated by their dif- ferent binding affinities for other glycan structures, which will also affect data interpretation [47]. Any structural assumptions always need to be verified by a complementary technique [42,43]. The same limita- tions apply for different antibody-binding profiles, particularly if the epitope is composed of peptide plus glycan. Nonspecific binding of antibodies is also com- mon [42]. It is surprising to note that the exact struc- tural epitope recognized by the widely used diagnostic commercial antibody against the O-glycosylated cancer antigen CA125 (MUC-16, marker of ovarian cancer) is not known. MS may also be used to determine the overall glyco- sylation profile of an intact purified glycoprotein [48–50]. This can provide a general picture of the different glycoforms on the protein, but yields no site information. However, such a profile is difficult to obtain with a highly O-glycosylated mucin-type pro- tein, owing to its extensive glycan heterogeneity and very large mass. Released O-glycan analysis – what we can do Mucin-type O-glycans are built from eight core struc- tures, many with the same monosaccharide residues in different linkages (Fig. 1) [51]. Most commonly, core 1 and core 2 glycans are found in humans. Core 1 gly- cans are small glycans that are often terminated with sialic acid, whereas core 2 glycans have the potential to be elaborated into larger glycans. Many of the core structures have the same mass, and linkage analysis is usually needed to differentiate them. The glycomic approach of releasing and characterizing the total com- plement of O-glycans from proteins provides informa- tion about the heterogeneity of the glycan species present in a sample, and can greatly assist in interpret- ing complex glycopeptide data from the same protein. There are several techniques being used at this time to globally release O-linked oligosaccharides. Fig. 1. The eight different reported core structures of mucin-type O-glycans. The linkage positions are illustrated by the line connect- ing the monosaccharides, and all linkages not labelled with a are b-anomers. As illustrated, many of the cores have the same mass. P. H. Jensen et al. Mucin-type O-glycosylation FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS 83 O-glycan release As there are no specific enzymes that release all O-linked glycans, chemical release methods need to be used. O-glycans can be released chemically from glyco- proteins either in solution or from samples immobilized on a poly(vinylidene difluoride) membrane. Reductive b-elimination performed using sodium borohydride in potassium or sodium hydroxide releases the O-glycans and reduces them simultaneously. This reduction of the terminal sugar protects them from peeling reactions (degradation of the released glycans), and is the most commonly used release method [52]. It is advisable to treat glycoprotein samples with N-glycosidase F before using this method, as N-linked glycans can also be par- tially released by reductive b-elimination conditions, and will complicate the subsequent interpretation. Reductive b-elimination, however, does not allow for subsequent fluorescent or colorimetric labelling (e.g. with 2-aminobenzamide, 1-phenyl-3-methyl-5-pyrazo- lone, or anthranilic acid) of the reducing terminus of O-glycans, as is used for N-glycan detection and quan- titation [53–56]. b-Elimination using hydrazine has been explored widely in an attempt to release O-glycans and retain the reducing end, without too much peeling of the glycans [57–59]. A nonreductive b-elimination method has also been described [60], and an alternative method of releasing the glycans by b-elimination in a mix of tetrahydroborate and tetradeuterioborate incor- porates a deuterium label in the reduced terminus for comparative quantitation [61]. Another approach, using the addition of a chemical tag during b-elimina- tion and Michael addition, yields side reactions and is not specific for mucin-type O-glycans [62]. These label- ling approaches are particularly useful for the fluores- cent quantification of the released O-glycans. It is, however, the belief of the authors that techniques involving derivatization of the reducing terminus of eliminated O-glycans have the potential to produce artefacts, destroy oligosaccharide modifications, and decrease sample yield, and that their use should there- fore be kept to a minimum. Separation of released O-glycans Several different chromatographic materials have been used to separate released, reduced O-glycans. Graphi- tized carbon has the remarkable capacity to separate different structural isomers of glycans that have the same composition [61,63,64]. This separation is based on size, linkages and ⁄ or branching, and allows a quick comparison of a large set of samples. Exoglycosidase digestions of the sample and ⁄ or tandem MS of the separated peaks can help to elucidate the structures. Another chromatographic material commonly used in the separation of glycans is primary amine-bonded sil- ica [61,65,66], and if separation of neutral and acidic glycans is desired, cation or anion exchange is a good choice [54,67,68]. For separation of hydrazine released, fluorescently labelled glycans, normal-phase chroma- tography is often used [69]. The separation of labelled as well as non-labelled O-glycans can be monitored either on-line via a detector (i.e. fluorescence, UV, or MS) or off-line (often larger scale), when fractions are collected and analysed separately. Detection of released O-glycans MS has become one of the preferred methods for both N-glycan and O-glycan analysis, owing to the sensitiv- ity and relative ease of use. MS and MS ⁄ MS analysis can be performed with both MALDI and ESI ioniza- tion, and there are advantages and disadvantages of both. For MALDI-MS analysis, glycan samples are often separated into neutral and acidic glycans, as the two have widely differing ionization properties. Anionic glycans do not respond well in positive ion mode MALDI-MS, whereas neutral glycans do not ionize as well in negative ion mode. Many laboratories perme- thylate the hydroxyl groups on the released glycans prior to MS analysis. Permethylation also methylates the carboxyl group of sialic acid, and can be used as a means of making all glycans neutral [70]. This approach has the added advantages of increasing the mass of the smaller O-glycans and stabilizing the sialic acids against loss for MALDI analysis, as well as directing the fragmentation of the glycans in MS ⁄ MS. Disadvantages are the increased sample manipulation and the possible loss of any modifications that may be present on the glycans, such as acetylation, sulfation, and phosphorylation, owing to the conditions of deriv- atization. Dihydroxybenzoic acid is the most commonly used matrix, and has been used in both negative and posi- tive ion mode MALDI-MS [54,65,67,68,71]. Other studies have used 3-aminoquinoline [68], dihydroxyace- tophenone [61] and ammonium citrate [54] matrix in the analysis of acidic glycans in negative ion mode. MALDI-MS is often used as a global glycan profiling technique, but unless the isomers are fractionated off-line, the approach does not give information on the possible compositional isomers, as they have the same m ⁄ z. In general, O-linked glycans are smaller and more diverse structures than N-linked glycans, and in MALDI-MS, where the matrix produces a lot of noise Mucin-type O-glycosylation P. H. Jensen et al. 84 FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS in the low-mass range, detection of the smaller O-gly- cans may be difficult. Released O-glycans can also be analysed by ESI-MS and MS⁄ MS, and this can result in specific diagnostic ions for specific structures [72]. This MS is often cou- pled with on-line LC separation. The authors favour this approach, using graphitized carbon chromatogra- phy, as it accomplishes isomeric separation and the simultaneous detection of both neutral and acidic gly- cans using a single chromatographic separation with negative ion mode ESI-MS detection [73–76]. Table 1 gives examples of the released mammalian O-mucin- type glycan masses and compositions that are typically detected with this approach. The masses listed are designed to introduce the novice glycoproteomic mass spectrometrist to masses that correspond to common released, reduced O-glycans detected in negative ion mode ESI-MS. It should be emphasized that each mass may represent several different structures with the same given composition. In most cases, extracted ion chro- matograms of the O-glycans separated by the graphi- tized carbon column will indicate whether more than one structure is present, as the isobaric isomers will elute at different retention times. Alternative methods of LC-ESI-MS ⁄ MS have been used by Royle et al. [77]; in these, normal-phase chro- matographic separation of 2-aminobenzamide-labelled O-glycans was achieved in positive ion mode. Graphi- tized carbon LC-ESI-MS ⁄ MS has also been used to separate isomers of permethylated oligosaccharide aldi- tols [78], but this approach was found to be best for the separation of released neutral O-glycans. However, permethylated neutral and acidic O-glycan isomeric alditols can be successfully separated and sequenced with high sensitivity by reversed-phase LC-ESI- MS ⁄ MS [79]. One of the major limitations of MS analysis of gly- can samples is that different component monosaccha- rides have the same mass. Hexoses such as Glc, galactose and mannose all have the same mass, and it is still only possible to determine the monosaccharide composition by acid hydrolysis of the oligosaccharides and separation by high-performance anion exchange chromatography with pulsed amperometric detection [80], with GC-MS [81], or by labelling the hydrolysed monosaccharide residues with different UV [82] or fluorescent tags [53–56]. Similarly, although MS ⁄ MS can give some information on specific glycan linkages, obtaining this information usually requires further experimentation with specific exoglycosidase digestion [69], linkage analysis by GC-MS [83], or NMR [65,66,68,80]. O-glycopeptide analysis – the remaining challenge The important cornerstone of glycoproteomics is assigning macroheterogeneity and microheterogeneity, i.e. assigning both the glycosylation sites and the dif- ferent glycoforms present on each site. Obtaining the whole picture is still the major challenge in the analysis of mucin-type O-glycosylation. Obtaining the glycopeptide Glycopeptides with O-linked glycans on a single site are easier to analyse than large N-glycosylated peptides, as they usually have smaller, less heterogeneous glycan structures attached. Mucin-like domains, however, are much more difficult, as they have numerous O-linked sites in very close proximity. As mentioned before, these domains are rich in Ser, Thr, and Pro, which are not the amino acids cleaved by the most commonly used proteases, such as trypsin, Lys-C, and chymotryp- sin. In fact, it is thought that one of the major functions of these domains and their glycans is to protect the pro- tein from proteolytic degradation. Often, nonspecific proteases have to be used, such as proteinase K [84] or pronase, either free [85] or immobilized [40]. These enzymes have been widely used in the analysis of N-linked glycosylation, where they produce a small amino acid tag with the intact glycans attached. Pronase has also been used for O-glycopeptide analysis [40], in which nonglycosylated peptides are completely digested and the remaining O-glycans are tagged with four to seven amino acids. One drawback to this Table 1. Some masses and compositions of commonly identified mucin-type released O-linked oligosaccharide alditols. Adapted from Thomsson et al. [137]. Commonly identified glycan masses a [M–H] Possible composition (reduced glycans, alditol form) 587.2 (Hex) 1 (HexNAc) 2 675.2 (Hex) 1 (HexNAc) 1 (NeuAc) 1 733.3 (Hex) 1 (HexNAc) 2 (deoxyhexose) 1 749.3 (Hex) 2 (HexNAc) 2 895.3 (Hex) 2 (HexNAc) 2 (Deoxyhexose) 1 966.3 (Hex) 1 (HexNAc) 1 (NeuAc) 2 1040.4 (Hex) 2 (HexNAc) 2 (NeuAc) 1 1041.4 (Hex) 2 (HexNAc) 2 (deoxyhexose) 2 1186.4 (Hex) 2 (HexNAc) 2 (deoxyhexose) 1 (NeuAc) 1 1187.5 (Hex) 2 (HexNAc) 2 (deoxyhexose) 3 1331.5 (Hex) 2 (HexNAc) 2 (NeuAc) 2 1332.5 (Hex) 2 (HexNAc) 2 (deoxyhexose) 2 (NeuAc) 1 a The masses are those of reduced glycans detected in negative mode carbon LC-ESI-MS. P. H. Jensen et al. Mucin-type O-glycosylation FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS 85 approach is that there is very heterogeneous cleavage of the amino acid backbone, and when this heterogeneity is added to the diversity of the attached glycans, it becomes difficult to interpret the mass spectra. Mirgorodskaya et al. [86] have used partial acid hydrolysis to successfully identify O-glycosylation sites in synthetic glycopeptides. They found that peptide bonds N-terminal to Asp, Ser and, occasionally, Thr and Gly were especially labile. They obtained good sequence coverage of most of the peptide, but signifi- cant hydrolysis of glycosidic bonds was also observed. Additionally, this method only works with known sequences of purified peptides, and cannot be applied to complex mixtures [87]. Enrichment of the glycopeptides after digestion of the protein improves their detection, as they are usu- ally less abundant than the nonglycosylated peptides in a digest, owing to glycan heterogeneity, and are also suppressed in the ionization process [88]. There are several general glycopeptide enrichment techniques, involving different chromatographic materials, such as Sepharose [89], boronic acid [90–92], hydrophilic liquid interaction chromatography [93–95], and graphite [84], whereas titanium dioxide [96] has been applied specifi- cally for the enrichment of sialylated glycopeptides. Enrichment of glycopeptides by oxidative hydrazide coupling of the sugars to a solid support [97,98] destroys the glycan, so this approach cannot be used for subsequent analysis of the oligosaccharide struc- tures on the glycopeptide. Similarly, methods that trim back glycans by partial deglycosylation (by successive incubation with exoglycosidases such as neuramini- dase, b-galactosidase and b-N-acetylhexosaminidase, or by chemical cleavage), or that produce glycoproteins in cell lines that have limited glycosylation machinery, provide a simpler protein glycosylation profile for site analysis [99,100], but do not give any information on the true glycosylation at each site. Site-specific assignment The methods currently available for determination of the glycan heterogeneity at specific sites of attachment of mucin-type O-glycans still have limitations. With N-glycans, where a site consensus sequence is known and only one or two sites are present on a tryptic pep- tide, it is relatively straightforward to determine the actual site of attachment. With mucin-type O-glycosyla- tion, there are often many Ser and Thr residues in close proximity within the glycopeptide that, in theory, could all be glycosylated. Therefore, sequencing of the peptide backbone with the glycans still attached is a prerequisite for unambiguous assignment and characterization of the heterogeneity of the occupied glycosylation sites. Ed- man sequencing was, for a long time, the only technique that allowed sequencing through glycopeptides to reveal the glycosylation sites, and, if performed on solid phase, gave partial information on the glycans attached [101]. MS has now emerged as the basic detector for pep- tide characterization. In the commonly used methods of collision-induced dissociation (CID) and IR multiph- oton dissociation fragmentation, glycans are detached from the amino acids by vibrational excitation, which mainly results in glycosidic fragmentation and some cross-ring cleavages. Although these data give some information on the branching and composition of the O-glycans on the peptide [102–104], there is hardly any fragmentation of the peptide backbone, and so no amino acid sequence information or glycan site identifi- cation is obtained [105]. In the last decade, new MS fragmentation techniques have emerged for potential use in the determination of mucin-type O-glycosylation sites, namely electron capture dissociation (ECD) [106,107] and electron transfer dissociation (ETD) [108,109]. ECD and ETD usually maintain labile modi- fications, owing to the high rate of amide bond cleav- age and the moderate amount of excess energy [110]. This leads to fragmentation of the peptide backbone with the modification still intact, opening the possibility of determining sites with the glycan still attached. Edman sequencing Edman sequencing can be used in two different ways to determine glycosylation sites. A regular protein Edman sequencer will sequence through a glycosylated peptide and leave a blank cycle for each glycosylated amino acid. Sparrow et al. [111] have exploited this, and local- ized six O-glycosylated sites out of 10 Ser and Thr resi- dues in a peptide. Intact glycoamino acids do not elute in the nonpolar solvents used in Edman chemistry, so immobilizing the glycopeptide on a membrane prior to sequencing was shown to allow for the use of polar eluting solvents and detection of glycoamino acids in a peptide sequence [101,112–116]. This promising tech- nique is limited by the amount of sample needed (pmol), the need for peptide purification, the need for sialic acid removal and, more importantly, the current limited availability of commercial protein sequencers. ECD/ ETD-MS Without the attached glycan To date, most published work has used ECD ⁄ ETD to determine the sites of protein O-phosphorylation Mucin-type O-glycosylation P. H. Jensen et al. 86 FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS [117,118]. Studies to determine the sites of O-glycosyla- tion on a protein have usually reduced the complexity by removing the glycans and tagging the Ser or Thr. This yields information about the glycosylation sites, but gives no information about the glycan heterogene- ity at the different sites. For example, treatment with sodium hydroxide removes O-glycans, leaving dehydro- alanine in place of the modified Ser, and dehydrobu- tyric acid in place of the modified Thr [119], and the sites of glycosylation are determined by the change in the resulting mass of the peptide. The same effect can be obtained with ammonia treatment, which needs less clean-up prior to analysis [120]. Variants of this method using different chemistries for better detection of deglycosylated Ser or Thr residues have been used [62,121]. The drawbacks to this approach can be non- specific dehydration of unmodified Ser and Thr resi- dues, and the inability to determine whether the Ser and ⁄ or Thr residues were modified by glycans or by other groups such as phosphate, which are b-elimi- nated in the same way. Czeszak et al. [122] have reported an improved method for site determination, using dimethylamine-catalysed b-elimination of the gly- cosylated site and employing a fixed-charge derivatiza- tion of the N-terminus of the peptide with a phosphonium group. With CID-MS⁄ MS, the fixed charge greatly improved the peptide fragmentation, leading to good sequence coverage and site identifica- tion [87]. The same laboratory has used the fixed- charge approach on synthetic peptides with a single GalNAc attached [122]. These methods all help to determine the sites of O-glycosylation, but have the limitation of ‘throwing away’ the glycan structure and heterogeneity information. With the attached glycan Since the introduction of ECD ⁄ FT ion cyclotron reso- nance MS fragmentation in 1998 by Zubarev et al. [107], several studies have been published on the use of this fragmentation technique in the analysis of mucin- type O-glycosylation. Mirgorodskaya et al. (1999) [105] identified multiple O-glycan sites in several synthetic peptides with ECD. Haselmann et al. (2001) later assigned multiple O-linked sites occupied by both neu- tral and acidic glycans on an MUC1 peptide with known sequence [123]. Kjeldsen et al. (2003) [110] located several O-glycosylated sites on bovine milk protein PP3, the sequence and sites for which were mostly known. Later, Renfrow et al. (2007) identified several mucin-type glycosylation sites on an IgA pep- tide after removal of acidic glycans. They experienced some difficulties in ECD fragmentation around the glycosylated sites, and speculated that it was the glycan itself that obstructed fragmentation [124]. This was previously also suggested by Hakansson et al. (2001) [125]. Alternatively, they suggest that it may be the structure of the gas-phase ion that inhibits fragmenta- tion [126], owing to either the glycosylation or a high level of Pro in the peptide. Recently, Sihlbom et al. (2009) [127] analysed the site-specific glycosylation in recombinant MUC1 by nanoLC-ECD-MS ⁄ MS. The peptide analysed contained only one GalNAc per site, and ECD successfully assigned one to five sites in the known peptide. Even with a single GalNAc substitu- ent, many different glycoforms of the peptide were identified. The authors observed that low-abundance glycoforms may have been missed, because the sensi- tivity of the technique is quite low. ECD fragmenta- tion is thus able to determine glycosylation sites and some glycan heterogeneity, especially if the peptide sequence is known. De novo sequencing and assign- ment is still difficult to achieve by this method. ETD ⁄ ion trap MS is the newest type of fragmenta- tion [108,128] to show promising results in mucin-type O-glycosylation site analysis. A limited number of studies have been performed so far. Wu et al. (2007) [109] performed a thorough study on O-glycopeptides with ETD fragmentation, and found that isolation and fragmentation of the charge-reduced (CR) species by CID (CR-CID) yielded additional product ions (c and z), particularly for larger m ⁄ z peptide ions (> 1000). A related method used supplemental activa- tion to enhance fragmentation of all ETD ⁄ ECD frag- ment ions [129]. According to Wu et al. (2007) [109] CR-CID of a single isolated CR species generates spectra that are cleaner and easier to interpret than a general hit with supplemental activation. Other studies support the finding of limited fragmentation informa- tion being obtained from ETD of precursor ions with m ⁄ z values larger than 1000 [129,130]. In addition, the low-resolution data from ion traps makes charge state assignments of both precursor and fragment ions diffi- cult. The newer OrbiTrap technology offers higher- resolution scanning in conjunction with ETD fragmen- tation. In general, the ETD ⁄ linear trap is useful for detection of ions if speed and sensitivity is desired, whereas the ETD ⁄ OrbiTrap can be used if resolution and accuracy is the aim [131,132]. As yet, it is not possible to have speed, sensitivity and high resolution together in ETD mode. ETD sequencing of a known glycopeptide with one O-glycosylation site [133] and on an O-GlcNAc-substituted glycopeptide with up to eight charged ions (H + ) [118,134] has been successful, but this analysis also required the sequence of the peptide to be known. P. H. Jensen et al. Mucin-type O-glycosylation FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS 87 The difficulty of site-specific analysis by ETD ⁄ ion trap MS is shown in the analysis of multiply O-man- nosylated peptides from human a-dystroglycan [135], which demonstrates the huge heterogeneity that exists in the glycosylation of these mucin-like domains. Recently, Perdivara et al. (2009) [136] successfully performed ETD on O-linked glycopeptides containing one and two glycosylation sites with both neutral and acidic glycans attached. This is the first study to actually perform de novo site characterization of O-glycosylated peptides. Conclusion Commonly, either the analysis of the O-glycosylation on a protein has been largely overlooked, or the glycans have been removed, trimmed or desialylated to facilitate analysis. We believe that if conclusions are to be drawn about protein function, or if O-linked glycoprotein bio- markers are to be discovered, we need to characterize the complete O-linked glycoprotein, including the com- position and structure of its O-glycans and the oligosac- charide structural heterogeneity at each occupied amino acid site. Most of the tools are now available to deter- mine the compositions and structures of the attached O-glycans and to identify some of the sites that may be occupied by them. The development of ECD ⁄ ETD-MS fragmentation may provide the final step in determining the diversity and extent of glycosylation at each site. The success of this new technique will depend on good sample preparation and new software development to help in interpreting the complex spectra that result. Acknowledgements P. H. Jensen was supported by the Danish Agency for Science, Technology and Innovation (grant 272-07- 0066). D. 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REVIEW ARTICLE Mucin-type O-glycosylation – putting the pieces together Pia H. Jensen, Daniel Kolarich and Nicolle H. Packer Department of. of the GalNAc to Ser ⁄ Thr. This diversity determines the density and site occu- pancy of the mucin-type O-glycosylation [11,12]. Attachment of the initial GalNAc occurs in the Golgi, to the. assigning both the glycosylation sites and the dif- ferent glycoforms present on each site. Obtaining the whole picture is still the major challenge in the analysis of mucin-type O-glycosylation. Obtaining