The use of mass spectrometry for the proteomic analysis of glycosylation

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1 Proteomics 2006, 6, DOI /pmic REVIEW The use of mass spectrometry for the proteomic analysis of glycosylation Willy Morelle, Kévin Canis, Frédéric Chirat, Valegh Faid and Jean-Claude Michalski Unité Mixte de Recherche CNRS/USTL 8576, Université des Sciences et Technologies de Lille 1, Villeneuve d Ascq Cedex, France Of all protein PTMs, glycosylation is by far the most common, and is a target for proteomic research. Glycosylation plays key roles in controlling various cellular processes and the modifications of the glycan structures in diseases highlight the clinical importance of this PTM. Glycosylation analysis remains a difficult task. MS, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of glycoconjugates. This review describes methodologies based on MS for detailed characterization of glycoconjugates in complex biological samples at the sensitivity required for proteomic work. Received: February 20, 2006 Revised: April 5, 2006 Accepted: April 5, 2006 Keywords: Glycomics / Glycoproteomics / Glycosylation / Diseases / Oligosaccharides 1 Introduction The development of approaches for measurement of the relative expression of proteins between two (or more) samples is an essential aspect of systems biology. Proteomics provides tools for the expression profiling of proteins and the elucidation of functional relationships among proteins [1 3]. The main focus in proteomic analyses is aimed at large-scale identification of proteins in a given biological sample. This large-scale protein analysis is useful for monitoring and identifying proteins involved in physiological changes in cells or organisms since it is the proteins, rather than the genes that execute them, which engage in biological activity [4]. Correspondence: Dr. Willy Morelle, Unité Mixte de Recherche CNRS/USTL 8576, Glycobiologie Structurale et Fonctionnelle, IFR 118, Université des Sciences et Technologies de Lille 1, Villeneuve d Ascq Cedex, France willy.morelle@univ-lille1.fr Fax: Abbreviations: DHB, 2,5-dihydroxybenzoic acid; ECD, electron capture dissociation; GlcNAc, N-acetylglucosamine; HCC, hepatocellular carcinoma; IRMPD, infrared multiphoton dissociation; Man6-P, mannose 6-phosphate; PSD, post-source decay; WGA, wheat germ agglutinin A Once the proteins are identified, the next obvious step is characterization of their PTMs, if such are present in the proteins of interest. PTM is an important feature of a proteome, frequently conveying a specific biological activity or role to a protein. Modifications can occur at a single or at multiple sites, often in varying forms. Among more than 100 different types of PTMs [5], each plays an important role. It has been claimed that up to 80% of mammalian cells contain PTMs [6], and a key challenge is to appreciate their role. Of all protein PTMs currently known, such as acetylation, biotinylation, famesylation, formylation, myristoylation, phosphorylation, and glycosylation, the last is by far the most common, and is found in both eukaryotes and prokaryotes [7, 8]. Since this PTM is the most common, glycosylation is a target for proteomic research. Unlike the core proteins, glycan chains are expressed as a set of variations on a core structure and are polydisperse in nature. For these reasons, glycosylation increases the complexity of protein molecules and complicates efforts to identify protein expression patterns that correlate with disease states. Glycan chains in glycoproteins play key functions in biological processes including embryonic development, the recognition of hormones as well as toxins, and cell-to-cell interactions. They have been found to participate in many biological processes, such as inter- and intracellular activities [9], coordination of immune functions [10], cell division processes, and protein regulations and interactions. Some gly-

2 3994 W. Morelle et al. Proteomics 2006, 6, coconjugates act as receptors of various molecules on the cell surface. Increasing reports show carbohydrates exhibiting a variety of important biological functions, such as anti-coagulants, adhesion ligands, immunomodulators, antigenic microbial recognition factors, and inflammatory response addressins, among others [11, 12]. Carbohydrates can have a profound influence on the physicochemical properties of glycoproteins. The folding, solubility, aggregation, and propensity of these proteins to degrade by protease activity can be affected by the presence or absence of carbohydrate chains. They may be needed to maintain the stability [13] or the thermal stability [14]. Glycans are implicated as ligands in recognition phenomena such as fertilization [15]. Glycosylation also determines the localization, activity, and function of proteins and thereby plays key roles in controlling various cellular processes. Four types of glycosylation are known: (i) the N-linked glycans which are attached to asparagine residues in the consensus sequence Asn-Xxx-(Ser, Thr) via an N-acetylglucosamine (GlcNAc) residue and Xxx can be any amino acid except proline; (ii) the O-linked glycans where the sugar is attached to serine or threonine; (iii) glycosylphosphatidylinositol anchors, which are attached to the carboxyl terminus of certain membrane-associated proteins; and (iv) finally C-glycosylation, which has been found attached to tryptophan residues in certain membrane-associated and secreted proteins [16]. In rare cases other amino acid residues, e.g. cysteine or lysine, may also be glycosylated [17]. The two most common forms, N- and O-glycosylation, are both characterized by branched 3-D structures which are highly diverse in form and size. The N-linked glycans all contain a common trimannosyl-chitobiose core with one or more antennae attached to each of the two outer mannose residues (Fig. 1). N-glycans are synthesized from a common precursor, which is processed by stepwise trimming and stepwise addition of new monosaccharide residues. This common core precursor is Glc 3 Man 9 GlcNAc 2 -PP-Dol, which is synthesized by an evolutionary highly conserved process in the ER [18 20]. Subsequent to its synthesis the core glycan is transferred en bloc onto Asn-Xxx-(Ser, Thr) acceptor sites of nascent polypeptide chains by the oligosaccharyltransferase complex [21, 22]. In rare cases other amino acid residues, such as cysteine residues, may also be glycosylated. The N- glycans can be classified into three subgroups on the basis of the nature and location of monosaccharide residues added to the core. Trimming by a-glucosidases and a-mannosidases without any subsequent glycosyl addition to the periphery results in glycans having the composition Man 5 9 GlcNAc 2. These glycans containing only mannose residues attached to the core structure are called high mannose type or oligomannose. The glycans that contain N-acetyllactosamine (Galb1 3/4GlcNAc) in their antennal region are called complex type. These N-glycans constitute the most abundant class of mammalian oligosaccharides. These structures are characterized by the presence of variable numbers of antennae, the biosynthesis of which is initiated by the addi- Figure 1. The major N-linked glycans. This scheme is designed to be indicative of some of the possible N-glycan structures. Symbols: h, galactose; n, N-acetylglucosamine; s, glucose; n, fucose; m, N-acetyl-neuraminic acid; d, mannose. tion of GlcNAc stubs to the two a-mannose residues of the core. In mammals, the antennae are usually elongated by the addition of b-gal. Antennae can be lengthened by the sequential addition of GlcNAc and Gal residues, resulting in tandem repeats of LacNAc. Rarely, b-galnac is added to the GlcNAc stubs in order to give GalNAcb1 4GlcNAc antennae [23 25]. The third subgroup hybrid type contains both mannose residue and N-acetyllactosamine attached to the trimannosyl chitobiose core. These N-glycans share structural features of the high mannose type and complex type. They usually retain two mannoses on the 6-arm of the trimannosyl core. Besides, the b-mannose of the core is also a possible site for GlcNAc attachment, and a GlcNAc residue attached to the 4-position of this mannose is referred to as a

3 Proteomics 2006, 6, Technology 3995 bisecting residue. The presence or absence of an a-fucosyl residue on the core mannose further adds to the structural diversity of oligosaccharides. Biosynthesis of complex-type structures is completed by a variety of capping reactions, the most important in mammals being sialylation and fucosylation. Sialic acid residues impart a net negative charge on an otherwise neutral glycan. A large body of evidence suggests that the negative charge of sialic acid-containing glycoconjugates is important in cell cell interactions. Capping sugars are usually a-linked, unlike the backbone residues, which are normally b-linked. The biosynthesis of O-glycans is a post-translational process that occurs in the Golgi apparatus requiring the sequential action of several membrane-bound glycosyltransferases. The first step is the transfer of an N-acetylgalactosamine residue onto a serine or a threonine residue of the protein backbone. The addition of GalNAc to a Ser/Thr residue is catalysed by uridine diphosphate (UDP)-GalNAc:- polypeptide N-acetylgalactosaminyltransferases (pp-galnac- Ts). In fact, there are more than 15 pp-galnac-ts in humans, and they probably cooperate in various aspects of protein O- glycosylation in a cell [26]. Therefore, in mammals, the initiating monosaccharide is normally N-acetylgalactosamine, although other monosaccharide units have also been described to be involved in an O-glycosidic linkage to hydroxyl amino acids. Most are rare, but some, such as the O-GlcNAc in cytosolic and nuclear proteins, are quite ubiquitous [27, 28]. Mannose-linked O-oligosaccharides have been described in brain glycoproteins [29] and in sheep dystroglycan [30]. Subsequent addition of galactose and/or N-acetylglucosamine by specific transferases leads to the formation of the common O-glycan core structures (Fig. 2). There are at least eight O-glycans core structures, four of which are particularly widespread in mammalian glycoproteins. These cores can be further elongated and terminated by the transfer of sialic acid or fucose residues. The structural diversity introduced through elongation of these basic structures with residues such as Gal, Fuc, GlcNAc, and NeuAc can lead to a large number of structural variants. The interest in glycosylation is also due to the implication of carbohydrates in many pathologic states, such as cancer [31 33], atherosclerosis, cystic fibrosis [34], and rheu- Figure 2. The eight O-oligosaccharide core types. matoid arthritis [35]. Altered glycosylation has been reported during several genetic diseases including leukocyte adhesion deficiency type II [36] and hereditary erythroblastic multinuclearity with a positive acidified serum lysis test (HEM- PAS) [37] and galactosaemia [38, 39]. Changes in N-linked glycosylation have long been associated with the development of disease [40 43]. Carbohydrate modifications can profoundly affect protein function. Their importance in disease is evident from a growing number of embryonic lethal phenotypes seen in knock-out mice with defects in glycoconjugate assembly or processing [44]. Carbohydrate moieties of glycoproteins, which are displayed on cell surface membranes, are modified during carcinogenesis and development [45], and hence a large-scale protein glycosylation analysis has become important. The modifications of the carbohydrate structures highlight the clinical importance of this PTM as an indicator or effector of pathologic mechanisms [46, 47]. Characterization of changes in the expression of multiple proteins provides a powerful strategy for investigating complex pathophysiological processes and designing drug therapies. Most of the proteins present in body fluids (such as serum, cerebrospinal fluid, urine, milk, saliva) are glycosylated. These proteins are also the most easily accessible for diagnostic and therapeutic purposes. Therefore, it is not surprising that many clinical biomarkers and therapeutic targets are glycoproteins. These include, for example, prostate-specific antigen in prostate cancer and CA125 in ovarian cancer. In order to address the question of disease-related glycosylation alteration, sensitive and fast strategies for the analysis of the glycosylation of proteins are required. In glycoproteomics, key structural issues include: protein identification, location of glycosylation sites, and evaluation of the glycan heterogeneity. Nowadays the identification of proteins, in the term of proteomics, is routinely performed in many laboratories. 2-DE coupled to MS is now a mature and well-established technique. On the other hand, glycosylation analysis is recognized as one of the main current challenges in proteomics. The past few years have seen important developments in glycosylation analysis. MS in combination with modern separation methodologies has become one of the most powerful and versatile techniques for structural analysis of glycans and glycopeptides. MS provides many advantages over traditional analytical methods, such as low sample consumption and high sensitivity. No structural technique can match MS for the range of structural problems that can be addressed and the complexity of samples that can be analysed successfully. Glycosylation analysis in proteomics context is still in the age of early development but the integration of compatible approaches for proteomics and glycoproteomics in terms of scale and sensitivity is a rapidly developing field [48]. This review covers techniques based on MS for detailed characterization of glycoproteins in complex biological samples, mapping glycosylation sites in complex mixtures. The techniques are briefly described together with methods for the

4 3996 W. Morelle et al. Proteomics 2006, 6, release of N- and O-linked glycans from glycoproteins separated in SDS-PAGE, and the analysis of the glycoproteins, glycopeptides, and glycans using MS. 2 Isolation and analysis of glycoproteins using MS 2.1 General aspects A proteomic analysis usually has two steps: (i) protein separation and (ii) protein identification which also includes the characterization of the PTMs. There exist two major proteomics approaches: (i) 2-DE for protein separation, followed by MALDI-TOF-MS for protein identification and (ii) 1- or 2- D LC for protein separation combined with ESI or MS/MS for protein identification. Glycoproteins typically exist as a diverse population of glycoforms carrying between one and several dozen different glycans in variable molar amounts at glycosylation sites with varying degrees of site occupancy. Moreover, the wide variety of positional and anomeric structures makes it possible for saccharides to form as many as distinct structures from as few as six different monosaccharide units [49]. Each glycosylated site may contain many different glycan structures leading to pronounced heterogeneity (microheterogeneity). For example, human erythrocyte CD59 has over 100 different oligosaccharide structures on a single glycosylation site [10, 50]. In addition, different sites may be only partially glycosylated (macroheterogeneity). Therefore, full characterization of glycoproteins is a major challenge and continues demanding the best out of analytical tools and methodologies. 2.2 Isolation of glycoproteins Glycoproteins of interest may be encountered as either soluble or membrane-bound molecules. Detergents must be included in the extraction buffer to yield membrane-bound glycoproteins. 2-DE does not resolve membrane-bound glycoproteins very well, mainly due to the solubilization difficulties encountered during the initial IEF step that hinders protein transfer into the second SDS-PAGE dimension [51]. Recent advances in non-gel methods using LC/MS have provided an alternative to gels and have been successfully applied for the characterization of membrane-bound proteins [52, 53]. For the extraction and fractionation of soluble glycoproteins, detergents are usually not necessary. However, the addition of some detergents can increase extraction yields and reduce the presence of contaminants. Generally, if necessary, glycoproteins can be purified by most conventional protein separation methodologies, including ion exchange, size exclusion, partition, hydrophobic interaction, dyeligand, and affinity chromatography. These chromatography techniques can be used to reduce the complexity of some samples [54]. This is particularly the case when proteomic analysis of human serum or human plasma should be performed [55 60]. Indeed, proteomic analysis of human serum or human plasma represents an extreme challenge due to the dynamic range of the proteins of interest. The great advantage of affinity selection methods is that they can target a specific structural feature of a PTM, rapidly select the fraction of the proteome with the targeted PTM, and provide substantial simplification of the mixture. An affinity chromatography using immobilized mannose 6-phosphate (Man6-P) receptor was recently used to characterize the human brain lysosomal proteome with the goals of establishing a reference map to investigate human diseases, such as lysosomal storage diseases, and to gain insights into the cellular function of the lysosome [61]. Proteins containing Man6-P, a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified and resolved using 2-DE. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins; 41 were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. Electrophoretic separations in gels such as SDS-PAGE can be used to reduce the complexity of some samples, to analyse glycoproteins, and can be useful for separating differently glycosylated isoforms. Stains based on oxidation of the sugar chains can be used to visualize these glycoproteins. Besides, this technique can be used for the analysis of membrane-bound glycoproteins, since lectin affinity chromatography, which is generally used for the concentration of glycoproteins, cannot be used for the insoluble fraction because of the presence of detergent in the solvent medium [62]. After separation, proteins are transferred by electroblotting onto a membrane for specific detection. The specific binding of diverse lectins to glycans can be used for detection and isolation of glycoproteins from human body fluids such as serum, milk, and saliva [63]. It can be particularly informative to compare the profiles of proteins in a complex sample with those revealed with the specific lectin, indicating which proteins are glycosylated and/or aberrantly glycosylated [62, 64]. 2.3 Lectin affinity chromatography Lectins constitute a group of proteins with unique affinities toward carbohydrate structures. Lectin affinity chromatography has now been developed for purification and concentration of glycoproteins and has become a powerful tool in glycoproteomics. The most commonly used technique involves a small column containing lectin attached to agarose-based material [65, 66], whereby the sample is loaded by using gravity-flow mode. After eliminating non-specific binding, glycoproteins are eluted by displacement from the column with an elution buffer which has the composition of the loading buffer plus a haptene saccharide. Various lectins can be used to isolate glycoproteins having distinct types of carbohydrate structures, e.g. galectins specific for LacNAc-con-

5 Proteomics 2006, 6, Technology 3997 taining glycans found exclusively in both N-glycans and O- glycans [67, 68], ConA which binds to high mannose-type and hybrid-type oligosaccharides with high affinity [69, 70], wheat germ agglutinin A (WGA) which recognizes N-acetylglucosamine and sialic acid residues, PNA specific to T-antigen found commonly in O-glycans [71], and Aleuria aurantia lectin (AAL) showing broad specificity for L-Fuc-containing glycans [72]. Identification by lectins may be misleading due to their different affinities of binding to similar structures. While glycoproteins can be differentiated on the basis on their different glycan moieties through lectin specificity, several cases of lectin affinity chromatography use lectins with broad specificity, such as Con A and WGA to isolate the entire pools of glycoproteins present in body fluids rather than specific structural types. The ability of different lectins (Con A, WGA, and jacalin) to recognize specific glycosylation motifs was used to develop a multi-lectin affinity system that can achieve a comprehensive capture of serum glycoproteins [73]. The jacalin lectin is known as a specific tool for capturing O-glycosylproteins, such as IgA1 [74], and its sugarbinding preference is well established for T (Galb1 3Gal- NAca) as well as Tn-antigens [75, 76]. The capture of glycoproteins was demonstrated to be specific, efficient and reproducible with this multi-lectin column. The results obtained with this affinity step indicated that about 10% of human serum proteins are glycosylated (w/w). Actually, 50% of the low-abundance serum proteins correspond to glycoproteins (if the top six abundant proteins are removed). Besides, this multi-lectin column can be used to screen serum proteins for glycosylation changes due to disease [77]. Using this multi-lectin affinity column followed by trypsin digestion and LC-MS/MS, 150 glycoproteins from human plasma and serum samples were isolated and characterized [78]. The multi-lectin column was useful for both capturing the glycoproteins as well as depleting non-glycosylated proteins. The potential of lectin affinity chromatography was recently used to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans [79]. Many biomolecular changes occurred during the development of hepatocellular carcinoma (HCC), including glycosylation [80 82]. The most notable change in glycosylation is an increase in the level of fucosylation of a-fetoprotein [83, 84]. In order to discover serum markers that can assist in the early detection of hepatitis B virus-induced liver cancer, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation was described [79]. A multi-lectin affinity (fucose recognizing lectins from Lens culinaris, Pisum sativum, and Vicia faba) was used to extract glycoproteins containing the glycan structures of interest and glycoproteins were resolved by 2-DE and identified by immunological or biochemical methods. Golgi Protein 73 (GP73) was found to be elevated and hyperfucosylated in woodchuck with HCC and in the serum of people with a diagnosis of HCC. More recently, using this targeted glycoproteomic methodology, Comunale et al. [85] identified 19 hyperfucosylated serum glycoproteins derived from patients diagnosed with hepatitis B virusinduced liver cancer. 2.4 Characterization of glycoproteins using MS MALDI-TOF-MS and ESI-MS are powerful tools for the characterization of intact glycoproteins [86 90]. To a variable extent, the intact glycoproteins can be resolved to their individual glycoforms by both methodologies if the glycoproteins correspond to small proteins (up to kda) and/or contain a limited number of glycan chains. If the mass of the protein is known, the glycan mass can be determined by the difference and the numbers of constituent monosaccharides in terms of hexose, deoxyhexose, acetamidodeoxyhexose, sialic acids can be elucidated since N-glycans and O-glycans contain a limited number of type of monosaccharide residues. With larger molecules, resolution of glycoforms becomes increasingly difficult. Two strategies are then possible: removal of the glycan, or cleavage of the peptide into smaller units. The measurement of protein molecular weight before and after removal of the attached glycans provides information on the state of glycosylation, even though the individual glycoforms may not be resolved [91]. The difference between the glycoprotein molecular weight obtained via MALDI-MS and the deduced molecular mass from the amino acid sequence can also provide information about the carbohydrate content of the glycoprotein [92]. ESI-MS can also provide analytically useful information for glycoproteins [93]. The resolution of the quadrupole instrument was insufficient to resolve the glycocomposition microheterogeneities of large glycoproteins in the molecular weight range of , but the average molecular weight of the glycoproteins can still be determined. As for the MALDI, the difference between this measured average molecular weight and the sequence molecular weight reflects the degree of PTMs in the protein. Claverol et al. [94] proposed a strategy combining the bottom up and top down approaches to characterize protein isoforms resulting from PTMs. This promising approach in proteomics is based on PAGE separation of protein isoforms, MS and MS n analyses of gel-eluted intact proteins using an IT mass spectrometer, and MS n analyses of proteolytic peptides. Protein isoforms up to 50 kda are extracted from polyacrylamide gels by passive elution using SDS, and SDS is removed using nanoscale hydrophilic phase chromatography. ESI-MS analysis of the intact protein isoforms is performed to determine the molecular mass and the number of PTMs. The presence of labile PTMs such as phosphorylations and glycosylations are confirmed by ESI-MS n analyses of the intact proteins. Finally, proteins are digested in solution, and MS/MS analyses of the modified peptides are performed to locate the modifications.

6 3998 W. Morelle et al. Proteomics 2006, 6, The ultrahigh resolution and sensitivity of ESI FT-ICR MS can be used to analyse intact molecules with molecular masses above 20 kda [95 97]. ESI FT-ICR MS has for the first time been exploited for the characterization of highly sialylated glycoproteins, using human a-1-acid glycoprotein as the model compound. Nagy et al. [98] have demonstrated that ESI FT-ICR MS is a useful tool for investigating intact, highly sialylated glycoproteins without the need of enzymatic or chemical digestion, derivatization, or preliminary chromatographic separation. 3 Analysis of glycopeptides 3.1 General aspects To determine the oligosaccharide site occupancy and sitespecific microheterogeneity over the entire polypeptide chain, characterization of the glycoforms representing each putative site is essential. Digestion with specific endoproteases is usually employed to separate the potential N-linked glycosylation sites on individual peptides. It is rather difficult to identify glycopeptides using MS in a complex protein digest. This is partly due to the low sensitivity of the detection of glycopeptides caused by site heterogeneity. Glycopeptide signals are often suppressed in the presence of other peptides, especially if the glycopeptides contain complex glycans with the negatively charged sialic acid moiety. For this reason, the peptide and glycopeptide mixture can be separated prior to MS/MS investigations. Significant progress has recently been made in the field of LC-MS-based methods for studying protein glycosylation [99]. The advantage of using ESI-MS for tryptic mapping of glycoproteins was first shown by Ling et al. [100]. The major advantage of this technique is that carbohydrate-specific ions can be selectively detected at high sensitivity during the chromatographic separation of complex digest mixtures [101]. Glycopeptides may be identified at the low picomole level by the appearance of oxonium ions such as m/z 204 (HexNAc), 163 (Hexose), 292 (sialic acid) and 366 (Hexose- HexNAc) that appear at high source-orifice potential [102, 103]. Such determinations can be performed on a single or triple quadrupole mass spectrometer equipped with an ESI source. The characteristic ions are produced by either increasing the orifice voltage in the ESI source or through CID within the collision cell of the triple quadrupole mass spectrometer. A triple quadrupole mass spectrometer furnishes higher selectivity, as it selectively detects only the parent ions that fragment in the second collision region of the triple quadrupole to produce the characteristic ions. 3.2 Enrichment methods Since glycopeptides often constitute a minor portion of a complex peptide mixture, differentiation between glycosylated and non-glycosylated peptides prior to LC-MS/MS analysis is essential. One way to deal with the complexity of the proteome in proteomics is to use affinity chromatography methods to select peptides with a common type of PTM. Several procedures for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content have been proposed recently. Lectin affinity chromatography is the most commonly used tool to remove non-glycosylated peptides and concentrate glycopeptides for proteomic purposes. However, it must be kept in mind that binding to specific glycan structures precludes the use of affinity chromatography with lectins for primary collection of glycopeptides with an unknown variety of glycan forms. A strategy allowing identification of glycoproteins in complex mixtures derived from either human blood serum or a cancer cell line has been proposed by Geng et al. [104]. The methodology involved the following steps: (i) reduction and alkylation, (ii) proteolysis of all proteins in the mixture with trypsin, (iii) affinity chromatographic selection of the glycopeptides with an immobilized lectin, (iv) fractionation by RPLC, (v) MALDI-TOF-MS of individual RPLC fractions, and (vi) peptide identification based on a database search. This approach targets specific classes of glycoproteins for identification. The types of glycoproteins analysed were: (i) N-type glycoproteins of known primary structure, (ii) N-type glycoproteins of unknown structure, and (iii) O-type glycoproteins glycosylated with a single N-acetylglucosamine. The limitations of the method are that it does not discriminate between glycoforms of proteins, the heterogeneity is not determined and that the approach is not very fast since individual RPLC fraction must be analysed using MALDI-TOF-MS. Zhang et al. [105] suggested a method for the selective isolation, identification and quantification of N-glycosyl peptides. The methodology involved the following steps: (i) glycoprotein oxidation which converts the cis-diol groups of carbohydrates to aldehydes, (ii) derivatization of the aldehydes with hydrazide groups immobilized on a solid support to form covalent hydrazone bonds, (iii) proteolysis of the immobilized glycoproteins with trypsin, (iv) isotope labelling of the a-amino groups of the immobilized glycopeptides with isotopically light or heavy forms of succinic anhydride, (v) release of the N-glycopeptides using PNGase F, (vi) identification and quantification of the peptides using mlc-esi MS/MS or mlc separation followed by MALDI MS/MS. This strategy was successfully applied to the analysis of plasma membrane proteins and proteins contained in human blood serum. This solid-phase capture for the peptides that contain N-linked carbohydrates in the intact protein was recently used to show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns [106]. A strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample was described by Kaji et al. [107]. This strategy, termed isotope-

7 Proteomics 2006, 6, Technology 3999 coded glycosylation-site-specific tagging (IGOT), involves the following steps: (i) affinity capture of glycoproteins by a lectin from complex biological mixtures, (ii) tryptic cleavage of the glycoproteins and affinity capture of glycopeptides by the same lectin, (iii) peptide-n-glycosidase F (PNGase F) digestion of the glycopeptides in H 2 18 O and (iv) analysis of the 18 O-tagged peptides by an integrated 2-D LC-MS/MS technology. This method was applied to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins and allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Using another approach, Fan et al. [108, 109] have also identified some glycoproteins in C. elegans. This approach involves the following steps: (i) membrane proteins are solubilized in guanidine HCl, precipitated, and digested with trypsin, (ii) affinity capture of glycopeptides by lectin, (iii) PNGase F digestion of the glycopeptides, (iv) analysis of the resulting peptides by MALDI quadrupole TOF (MALDI-Q-TOF) MS. Together, these studies have identified 304 proteins containing N-glycans in C. elegans [107, 109]. The identification of these glycoproteins and characterization of their N-linked glycosylation sites are major steps toward understanding the in vivo roles played by N-glycans. Recently, Bunkenborg et al. [110] selected the glycoproteins from human serum by an initial lectin chromatography step and the glycoproteins were digested with endoproteinase Lys-C. Endoproteinase Lys-C enables digestion of proteins under denaturing conditions such as in the presence of 8 M urea. This facilitates digestion of some proteins, particularly those assembled in complexes. Glycosylated peptides were then concentrated by a second lectin chromatography step. The glycan components were removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line RPLC MS. Using two different lectins, ConA and WGA, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified. Qiu and Regnier [111] described a simple and rapid method based on serial lectin affinity chromatography (SLAC) for fractionation and comparison of glycan site heterogeneity on glycoproteins derived from human serum. The analytical protocol is based on glycopeptide selection from tryptic digests with serial lectin affinity chromatography, quantification with global internal standard technology, fractionation of deglycosylated peptides with RP chromatography, and peptide sequencing with a QSTAR quadrupole TOF mass spectrometer. Fractionation of complex tri- and tetra-antennary N-linked glycoforms from biantennary N- linked glycoforms bearing terminal sialic acid residues was achieved using a set of serial lectin columns with immobilized Sambucus nigra agglutinin and ConA. These two fractions from the affinity selection were differentially labelled, mixed, and then deglycosylated with the enzyme PNGase F. The deglycosylated sample was further fractionated by RP chromatography and analysed by ESI-MS. This proteomescale method can recognize and quantify differences or changes in the degree of branching between sialic acid-bearing glycan isoforms from specific glycosylation sites on proteins. Liu et al. [112] proposed an approach for the analysis of human plasma N-glycoproteome using a combination of immunoaffinity subtraction and glycoprotein capture. Six high-abundance plasma proteins albumin, IgG, antitrypsin, IgA, transferrin, and haptoglobin that constitute approximately 85% of the total protein mass of human plasma were simultaneously removed using a immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin, and digested using trypsin. The bound N-linked glycopeptides were released using PNGase F and the deglycosylated peptides were analysed by LC-MS-MS. In total, 2053 different N-glycopeptides were identified, covering 303 non-redundant N- glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance glycoproteins. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, a novel approach was proposed by Uematsu et al. [113] for focused proteomics. By using the potential of the MALDI-LIFT-TOF/TOF, this approach can provide information both on peptide sequence and glycan structure for the analysis of glycopeptides [114]. After characterizing both qualitatively and quantitatively the N-glycosylation profiles of the tissues, glycoproteins were digested with trypsin and glycopeptides were affinity captured using lectin chromatography. Fifteen glycoproteins with 19 N-glycosylation sites that carry high mannose-type glycans were identified by off-line LC-MALDI-TOF/TOF. This approach provides not only the identification of glycoproteins carrying particular glycoforms but also the determination of the N-glycosylation sites and the relative quantities of the microheterogeneous glycoforms present at each N-glycan binding site (Fig. 3). To address the effective isolation of trace glycoproteins from biological samples, future efforts need to be directed toward miniaturization of lectin-based procedures. Recently, Madera et al. [115] described an approach for the utilization of lectins to on-line analytical schemes and the investigations of glycoproteins at microscale. The authors demonstrated that silica-based lectin microcolumns provide a suitable alternative to agarose materials as an effective and reproducible means of on-line preconcentration of glycoproteins and glycopeptides from complex biological materials at trace levels. Using two different lectin preparations (Canavalia ensiformis and Sambucus nigra), the analytical benefits of the surface attachment such as coverage uniformity, binding capacity, trapping reproducibility, quantitative desorption for MS analysis, and enhanced measurement selectivity in glycoproteomics were demonstrated using model glycoproteins. The described analytical

8 4000 W. Morelle et al. Proteomics 2006, 6, Figure 3. Tandem mass spectrum derived from TOF/TOF of the [M1H] 1 precursor, m/z The amino acids are represented in their single letter codes. The sequence identification of the peptide and database search showed that this glycopeptide was derived from cathepsin L with the sequence of YYHGELSYLNVTR. The signal for the deglycosylated peptide is marked with an asterisk. 0,2X, ring fragmentation of the innermost GlcNAc [113]. (Reproduced by permission of the American Society for Biochemistry and Molecular Biology.) systems are amenable to the applications aiming at fractionation of complex glycopeptide mixtures and determination of the sites of glycosylation. All these approaches demonstrated that lectin affinity chromatography is a powerful tool for glycoproteomics. However, since lectins target specific oligosaccharide structures, lectin affinity chromatography cannot be used to prepare a glycopeptide pool suitable for characterization of total glycan heterogeneity [116, 117]. Wada et al. [118] proposed a method utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides for the isolation of tryptic glycopeptides. The enriched glycopeptide mixture can be directly analysed by MS for oligosaccharide structures at individual glycosylation sites. Both peptide and oligosaccharide structures were elucidated by multiple-stage MS/MS (MSn) of the ions generated by MALDI. The strategy, isolation of glycopeptides followed by MSn analysis, efficiently characterized the structures of b 2 - glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins. Recently, the glycopeptide enrichment method was improved in recovery, and applied to the analysis of site-specific N- and O- glycans of plasma and cellular fibronectin isoforms [119]. Larsen et al. [120] described a method that allows extensive characterization of a small amount of gel-separated N-linked glycoprotein. It uses a two-step proteolytic digestion combined with purification of the glycopeptides by sequential use of microcolumns packed with RP resin and graphite powder. The method allows protein identification, glycosylation site mapping, and partial structure analysis of the attached glycans. The method is fast and sufficiently sensitive to allow characterization of glycoproteins in gel-based proteomics. A large variety of nuclear and cytoplasmic proteins have been shown to be post-translationally modified by the addition of single N-acetylglucosamine moieties to serine and threonine residues [28, 121, 122]. Interest in this modification is increasing as evidence accumulates that it is an abundant and transient modification that is dynamic and responsive to cellular stimuli [ ]. The enzyme catalyzing O-GlcNAc modification (O-GlcNAc transferase) is essential for cell viability in mammals [124]. Efforts to identify O-GlcNAc-modified proteins have been challenged by the difficulty of detecting this modification in vivo. There is no consensus sequence for sites of modification [128]. In many instances, the sites of GlcNAcylation and phosphorylation are localized to the same or neighbouring residues [ ]. Besides, the stoichiometry of modification is often extremely low. O-GlcNAc is also a labile modification and this characteristic causes some problems for the localization of this modification. In most MS/MS experiments, O-GlcNAc is lost before the peptide itself fragments, preventing the localization of the glycosylation site. However, Chalkley and Burlingame [132, 133] have shown that site of O-GlcNAcylation can be identified directly using Q-TOF MS under certain circumstances. For the enrichment of low-abundance O-GlcNAc species from complex mixtures, several powerful approaches have been reported. Wells et al. [134] proposed an MS-based method for the identification of sites modified by O-GlcNAc that relies on mild b-elimination. The method is based on a mild b-elimination followed by Michael addition of DTT (BEMAD) or biotin pentylamine to tag O-GlcNAc sites. The tag allows for enrichment via affinity chromatography and is more stable during CID than the O-GlcNAc it replaces, facilitating mapping of modification sites (Fig. 4). The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on synapsin I purified from rat brain. An immunoaffinity and enzymatic strategy allows the discrimination between O-GlcNAc and phosphorylation sites with the use of BEMAD. Since the BEMAD methodology requires several controls to establish whether a peptide contains a phosphate or O-GlcNAc, a chemoenzymatic approach has recently been pro-

9 Proteomics 2006, 6, Technology 4001 Figure 4. DTT replacement of O-GlcNAc through BEMAD is stable during MS/MS, allowing for identification of the peptide and the DTTmodified residue. BEMAD was performed on the peptide PSVPVS(O-GlcNAc)GSAPGR, and the sample was analysed by nanospray LC-MS/ MS. (A), MS/MS spectrum from CID of a precursor ion selected at [M12H]2 1. All theoretical b and y ions are indicated by dashed lines. (B), interpretation of MS/MS data in A by Turbosequest search against the Owl database allowing for addition of Da to serine (*) or threonine (#) correctly identifies the peptide PSVPVS(DTT)GSAPGR. (C), b and y ion fragments correctly interpreted are shown in bold. Both the b and y ions ending at the DTT-modified serine are present, making assignment of the site of modification unambiguous [134]. (Reproduced by permission of the American Society for Biochemistry and Molecular Biology.) posed by Khidekel et al. [135, 136] that exploits an engineered galactosyltransferase enzyme to selectively label O-GlcNAc proteins with a ketone-biotin tag. After proteolytic cleavage, biotin-tagged O-GlcNAc peptides are purified using avidin affinity chromatography and analysed using nanoscale RP-HPLC-MS-MS. The tag permits enrichment of low-abundance O-GlcNAc peptides from complex mixtures and localization of the modification to short amino acid sequences. Using this approach, Khidekel et al. identified 25 O-GlcNAcglycosylated proteins from the brain. More recently, an LC-MS-MS coupled proteomic approach was reported for measuring changes in both expression and low-abundance serine/threonine PTMs. The method involves differential isotopic labelling by Michael addition with either light DTT (d0) or deuterated heavy DTT under conditions which discriminate between derivatization of O-phosphate and O-GlcNAc, or non-specific residues. After enrichment of the modified peptides by thiol chromatography, peptides were analysed by LC-MS-MS on a QSTAR Pulsar mass spectrometer. This approach provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping was achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis [137]. 3.3 MS of glycopeptides As we have seen for most of the approaches described so far, a commonly used strategy is to remove N-glycans with PNGase F before the MS analysis [90, 138, 139]. Although very useful for the identification of the glycoprotein and the localization of the glycosylation site, this strategy does not allow site-specific glycosylation profiling. Glycopeptides are usually analysed by MALDI-TOF-MS (Fig. 5) or by electrospray MS [ ]. Further analysis by MS/MS is mostly performed using electrospray instruments with a quadrupole and a TOF analyser, applying CID. However, the most frequently observed fragmentation is that corresponding to the loss of the glycan, leaving the site of attachment unmodified. Therefore, the fragmentation of glycosylated peptides, in most cases, provides information on the glycan moiety, but not on the attachment site. Because of these drawbacks, the potential of the recently introduced MALDI-TOF/TOF-MS instrument has been explored for the analysis of N-glycosylated peptides, using horseradish peroxidase [147]. Two different types of cleavage were observed in the TOF/TOF fragmentation spectra: Firstly, cleavages of peptide bonds yielded fragments with the attached N-glycans staying intact, thus revealing infor-

10 4002 W. Morelle et al. Proteomics 2006, 6, Figure 5. MALDI mass spectra of a recombinant human thyroid stimulating hormone glycopeptide before (A) and after treatment with a-neuraminidase (B); a-neuraminidase, and b-galactosidase (C); a-neuraminidase, b-galactosidase, and b-n-acetylhexosaminidase (D) [142]. (Reproduced by permission of John Wiley and Sons.) mation on peptide sequence and glycan attachment site. The complete retention of the glycan moiety resulted in spectra that were rather easy to interpret. Secondly, fragmentation of the glycan moiety was characterized by cleavage of glycosidic bonds as well as a 0,2 X-ring fragmentation of the innermost N-acetylglucosamine of the chitobiose core. Therefore, MALDI-TOF/TOF-MS spectra of N-glycosylated peptides provided information on the peptide sequence, the glycan attachment site, and the glycan structure. FT-ICR-MS is a method which provides high mass resolution, mass accuracy, and MS/MS capabilities simultaneously [148]. FT-ICR-MS offers two alternative fragmentation techniques, electron capture dissociation (ECD) [149, 150] and infrared multiphoton dissociation (IRMPD) [151, 152], both of which have been employed to selectively investigate protein glycosylation [ ]. ECD is a soft fragmentation technique that cleaves the amino acid backbone without removing the PTM [ ], often resulting in near complete sequence coverage and localization of the PTM (Fig. 6). Hakansson et al. [159] demonstrated that ECD in combination with IRMPD could be a powerful tool for the structural analysis of intact N-glycopeptides. Strictly complementary behaviour was observed for the two peptide fragmentation techniques. The fragment ions from the ECD analysis yielded a peptide sequence tag of six amino acid residues with no observed loss of glycans. In contrast, IRMPD provided abundant fragment ions, primarily through dissociation at glycosidic linkages permitting structural information of the glycan to be acquired. The monosaccharide composition and the presence of three glycan branch sites could be determined. This permits obtaining structural information about both the peptide and the saccharide with no release of the glycan. Recently, it was demonstrated that dissociation resulting from ion/ion electron transfer (ETD) in a quadrupole ion trap mass spectrometer, in combination with CID, can provide similar information about both peptide and glycan structures for glycopeptides [160]. The use of MALDI-TOF/TOF-MS in glycopeptide analysis results in peptide-backbone cleavage ions with no fragmentation of the glycan [147]. Thus MALDI-TOF/TOF- MS resembles FT-ICR-MS with tandem ECD, where peptide fragmentation occurs faster than the glycan loss allowing the localization of the glycosylation sites. Both techniques provide detailed sequence and site-of-attachment data for glycosylated proteins. This is in contrast to CID fragmentation of [M1H] 1 ions of the peptide moiety of glycopeptides using electrospray instruments with a quadrupole and a TOF analyser, which are characterized by abundant ions from glycosidic bond and very lowabundant ions from peptide-bond fragmentation [161]. However, several works have demonstrated that this mass spectrometer can be used to characterize O-glycosylation sites [162, 163].

11 Proteomics 2006, 6, Technology 4003 Figure 6. ECD of 51 of the glycosylated zonadhesin peptide with M r 3074 (five GalNAc groups) isolated from the Gal- NAc-T1 generated glycoform mixture. Two z? fragment ions (z7? and z14? ) are accompanied by a loss of one glycan out of the total two and five, respectively [153]. (Reproduced by permission of the American Chemical Society.) 4 Glycosylation analysis of gel-separated proteins 4.1 General aspects 2-DE is an analytical technique that simultaneously separates thousands of proteins and allows comparative protein profiling between different crude biological samples. Although labour intensive, this technique is still the predominant method for protein profiling. Long IPG strips with narrow ph ranges have been designed to increase the resolution of 2-DE separation. Indeed, when narrow pi range IEF gels are employed, the 2-DE technique has an unparalleled resolving capability for fractionating complex protein mixtures [164]. Modified forms of the same protein can be separated into several spots on the 2-DE gel. For example, glycosylated proteins give rise to a characteristic train of spots with different pi and apparent M r. For these reasons, 2-DE is the preferred method for separating the glycoforms of proteins. The expression of these different glycoforms may be useful as a diagnostic marker [165]. Moreover, the staining intensity of 2-DE spots roughly reflects the protein amount, especially for similar proteins; therefore, this method also provides information about the relative proportion of the various modified states. After separating the proteins by 2-DE, the resultant spots can be cut, destained, digested by trypsin, and analysed by MALDI-TOF-MS. Tools such as ExPASy, that scan the different protein databases available, allow the identification of the protein based on its pi, M r, and the masses of the different peptides generated from the trypsin digestion of this protein [ ]. The pi and M r can be estimated from the location of the spot on the gel. This is the most widely used approach in proteomics using MS and is referred to as bottom up strategy. MS has now reached a level in which the structural analysis of glycoproteins and their glycans can be undertaken on a gel-separated protein. MALDI and ESI are the preferred MS techniques for proteomic work providing sensitivity in the low picomole to high femtomole range. There are a number of analytical techniques that have been shown to obtain data from gel-separated glycoproteins. These techniques mainly focus on the determination of glycan profiles. 4.2 Global glycosylation analysis Global glycosylation corresponds to the glycosylation profile of an isolated glycoprotein. Küster et al. [171] developed a rapid and sensitive method for profiling and sequencing of N-glycans from standard glycoproteins following their release directly from within SDS gels using PNGase F. After digestion of PNGase F, the glycans were extracted with water and ACN and purified with a three-bed microcolumn packed with AG-3, AG-50 and C 18 resins. Global profiling of the neutral glycans from pmol of glycoproteins was obtained using MALDI-TOF-MS and normal-phase HPLC following fluorescent labelling with 2-aminobenzamide. For sialylated glycans, it was necessary to stabilize these glycans for the MALDI-TOF-MS analysis by esterification of the carboxylic acid group on the sialic acids [172]. Besides, this modification allows their clean-up using the three-bed microcolumn. After extraction of the glycans, the identity of the protein can be confirmed by in-gel trypsin digestion followed by MALDI-TOF-MS mass mapping. This in-gel release method was extended to sulphated glycans by Wheeler and Harvey [173]. Packer et al. [174] reported techniques for the analysis of glycans released from glycoproteins that have been electroblotted to a PVDF membrane after 1-D and 2-D preparative gel electrophoresis. The N-glycans were removed using PNGase F and O-glycans were chemically released and sepa-