Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation

Glycosylation is an important post-translational modification. Analysis of glycopeptides is difficult using collision-induced dissociation, as it typically yields only information about the glycan structure, without any peptide sequence information. We demonstrate here how a 3D-quadrupole ion trap, using the complementary techniques of collision induced dissociation (CID) and electron-transfer dissociation (ETD), can be used to elucidate the glycan structure and peptide sequence of the N-glycosylated peptide from a fractionated tryptic digest of the lectin from the coral tree, Erythina cristagalli. CID experiments on the multiply protonated glycopeptide ions yield, almost exclusively, cleavage at glycosidic bonds, with little peptide backbone fragmentation. ETD reactions of the triply charged glycopeptide cations with either sulfur dioxide or nitrobenzene anions yield cleavage of the peptide backbone with no loss of the glycan structure. These results show that a 3D-quadrupole ion trap can be used to provide glycopeptide amino acid sequence information as well as information about the glycan structure.     

Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos

Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resolution and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive experimental comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale analysis of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the analysis of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissociation (HCD) and ETD can improve the average Mascot score.     

PTM MarkerFinder,a software tool to detect and validate spectra from peptides carrying post‐translational modifications

Mass spectrometry (MS) analysis of peptides carrying post‐translational modifications is challenging due to the instability of some modifications during MS analysis. However, glycopeptides as well as acetylated, methylated and other modified peptides release specific fragment ions during CID (collision‐induced dissociation) and HCD (higher energy collisional dissociation) fragmentation. These fragment ions can be used to validate the presence of the PTM on the peptide. Here, we present PTM MarkerFinder, a software tool that takes advantage of such marker ions. PTM MarkerFinder screens the MS/MS spectra in the output of a database search (i.e., Mascot) for marker ions specific for selected PTMs. Moreover, it reports and annotates the HCD and the corresponding electron transfer dissociation (ETD) spectrum (when present), and summarizes information on the type, number, and ratios of marker ions found in the data set. In the present work, a sample containing enriched N‐acetylhexosamine (HexNAc) glycopeptides from yeast has been analyzed by liquid chromatography‐mass spectrometry on an LTQ Orbitrap Velos using both HCD and ETD fragmentation techniques. The identification result (Mascot .dat file) was submitted as input to PTM MarkerFinder and screened for HexNAc oxonium ions. The software output has been used for high‐throughput validation of the identification results.     

De novo peptide sequencing using CID and HCD spectra pairs

In tandem mass spectrometry (MS/MS), there are several different fragmentation techniques possible, including, collision‐induced dissociation (CID) higher energy collisional dissociation (HCD), electron‐capture dissociation (ECD), and electron transfer dissociation (ETD). When using pairs of spectra for de novo peptide sequencing, the most popular methods are designed for CID (or HCD) and ECD (or ETD) spectra because of the complementarity between them. Less attention has been paid to the use of CID and HCD spectra pairs. In this study, a new de novo peptide sequencing method is proposed for these spectra pairs. This method includes a CID and HCD spectra merging criterion and a parent mass correction step, along with improvements to our previously proposed algorithm for sequencing merged spectra. Three pairs of spectral datasets were used to investigate and compare the performance of the proposed method with other existing methods designed for single spectrum (HCD or CID) sequencing. Experimental results showed that full‐length peptide sequencing accuracy was increased significantly by using spectra pairs in the proposed method, with the highest accuracy reaching 81.31%.     

Effectiveness of CID, HCD, and ETD with FT MS/MS for degradomic-peptidomic analysis: comparison of peptide identification methods

We report on the effectiveness of CID, HCD, and ETD for LC-FT MS/MS analysis of peptides using a tandem linear ion trap-Orbitrap mass spectrometer. A range of software tools and analysis parameters were employed to explore the use of CID, HCD, and ETD to identify peptides (isolated from human blood plasma) without the use of specific "enzyme rules". In the evaluation of an FDR-controlled SEQUEST scoring method, the use of accurate masses for fragments increased the number of identified peptides (by ~50%) compared to the use of conventional low accuracy fragment mass information, and CID provided the largest contribution to the identified peptide data sets compared to HCD and ETD. The FDR-controlled Mascot scoring method provided significantly fewer peptide identifications than SEQUEST (by 1.3-2.3 fold) and CID, HCD, and ETD provided similar contributions to identified peptides. Evaluation of de novo sequencing and the UStags method for more intense fragment ions revealed that HCD afforded more contiguous residues (e.g., ≥ 7 amino acids) than either CID or ETD. Both the FDR-controlled SEQUEST and Mascot scoring methods provided peptide data sets that were affected by the decoy database used and mass tolerances applied (e.g., identical peptides between data sets could be limited to ~70%), while the UStags method provided the most consistent peptide data sets (>90% overlap). The m/z ranges in which CID, HCD, and ETD contributed the largest number of peptide identifications were substantially overlapping. This work suggests that the three peptide ion fragmentation methods are complementary and that maximizing the number of peptide identifications benefits significantly from a careful match with the informatics tools and methods applied. These results also suggest that the decoy strategy may inaccurately estimate identification FDRs.     

Evidence for a site-specific fucosylation of N-linked oligosaccharide of immunoglobulin A1 from normal human serum

Glycopeptides containing the N-linked oligosaccharide from human serum IgA1 were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). Two glycopeptides, GP1 and GP2, prepared from the endoproteinase Asp-N digest of the IgA1 heavy chain, were derived from the CH2 domain (N-glycan site at Asn263) and the tailpiece portion (N-glycan site at Asn459), respectively. The structure of the attached sugar chain was deduced from the mass number of the glycopeptide and confirmed by a two-dimensional mapping technique for a pyridylaminated oligosaccharide. GP1 was composed of two major components having a fully galactosylated bianntena sugar chain with or without a bisecting N-acetylglucosamine (GlcNAc) residue. On the other hand, the GP2 fraction corresponded to the glycopeptides having a fully galactosylated and fucosylated bianntena sugar chain partly bearing a bisecting GlcNAc residue. Thus, the site-specific fucosylation of the N-linked oligosaccharide on the tailpiece of the 1 chain became evident for normal human serum IgA1.     

N-Glycan structure annotation of glycopeptides using a linearized glycan structure database (GlyDB)

While glycoproteins are abundant in nature, and changes in glycosylation occur in cancer and other diseases, glycoprotein characterization remains a challenge due to the structural complexity of the biopolymers. This paper presents a general strategy, termed GlyDB, for glycan structure annotation of N-linked glycopeptides from tandem mass spectra in the LC-MS analysis of proteolytic digests of glycoproteins. The GlyDB approach takes advantage of low-energy collision-induced dissociation of N-linked glycopeptides that preferentially cleaves the glycosidic bonds while the peptide backbone remains intact. A theoretical glycan structure database derived from biosynthetic rules for N-linked glycans was constructed employing a novel representation of branched glycan structures consisting of multiple linear sequences. The commonly used peptide identification program, Sequest, could then be utilized to assign experimental tandem mass spectra to individual glycoforms. Analysis of synthetic glycopeptides and well-characterized glycoproteins demonstrate that the GlyDB approach can be a useful tool for annotation of glycan structures and for selection of a limited number of potential glycan structure candidates for targeted validation.     

On-line LC-MS approach combining collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced species for the trace-level characterization of proteins with post-translational modifications

We have expanded our recent on-line LC-MS platform for large peptide analysis to combine collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced (CRCID) species derived from ETD to determine sites of phosphorylation and glycosylation modifications, as well as the sequence of large peptide fragments (i.e., 2000-10,000 Da) from complex proteins, such as beta-casein, epidermal growth factor receptor (EGFR), and tissue plasminogen activator (t-PA) at the low femtomol level. The incorporation of an additional CID activation step for a charge-reduced species, isolated from ETD fragment ions, improved ETD fragmentation when precursor ions with high m/z (approximately >1000) were automatically selected for fragmentation. Specifically, the identification of the exact phosphorylation sites was strengthened by the extensive coverage of the peptide sequence with a near-continuous product ion series. The identification of N-linked glycosylation sites in EGFR and an O-linked glycosylation site in t-PA were also improved through the enhanced identification of the peptide backbone sequence of the glycosylated precursors. The new strategy is a good starting survey scan to characterize enzymatic peptide mixtures over a broad range of masses using LC-MS with data-dependent acquisition, as the three activation steps can provide complementary information to each other. In general, large peptides can be extensively characterized by the ETD and CRCID steps, including sites of modification from the generated, near-continuous product ion series, supplemented by the CID-MS2 step. At the same time, small peptides (e.g., 相似文献   

The N-linked oligosaccharides of aminopeptidase N from Manduca sexta: site localization and identification of novel N-glycan structures.

Mass spectrometric studies on the N-linked glycans of aminopeptidase 1 from Manduca sexta have revealed unusual structures not previously observed on any insect glycoprotein. Structure elucidation of these oligosaccharides was carried out by high-energy collision-induced dissociation (CID) using a matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometer. These key experiments revealed that three out of the four N-linked glycosylation sites in this protein (Asn295, Asn623 and Asn752) are occupied with highly fucosylated N-glycans that possess unusual difucosylated cores. Cross-ring fragment ions and 'internal' fragment ions observed in the CID spectra, showed that these fucoses are found at the 3-position of proximal GlcNAc and at the 3-position of distal GlcNAc in the chitobiose unit. The latter substitution has only been previously observed in nematodes. In addition, these core structures can be decorated with novel fucosylated antennae composed of Fucalpha(1-3)GlcNAc. Key fragment ions revealed that these antennae are predominantly found on the upper 6-arm of the core mannose. The paucimannosidic N-glycan (Man(3)GlcNAc(2)), commonly found on other insect glycoproteins, is the predominant oligosaccharide found at the remaining N-glycosylation site (Asn609).     

Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment

Mass spectrometry-based studies of proteins that are post-translationally modified by O-linked β-N-acetylglucosamine (O-GlcNAc) are challenged in effectively identifying the sites of modification while simultaneously sequencing the peptides. Here we tested the hypothesis that a combination of high-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD) could specifically target the O-GlcNAc modified peptides and elucidate the amino acid sequence while preserving the attached GlcNAc residue for accurate site assignment. By taking advantage of the recently characterized O-GlcNAc-specific IgG monoclonal antibodies and the combination of HCD and ETD fragmentation techniques, O-GlcNAc modified proteins were enriched from HEK293T cells and subsequently characterized using the LTQ Orbitrap Velos ETD (Thermo Fisher Scientific) mass spectrometer. In our data set, 83 sites of O-GlcNAc modification are reported with high confidence confirming that the HCD/ETD combined approach is amenable to the detection and site assignment of O-GlcNAc modified peptides. Realizing HCD triggered ETD fragmentation on a linear ion trap/Orbitrap platform for more in-depth analysis and application of this technique to other post-translationally modified proteins are currently underway. Furthermore, this report illustrates that the O-GlcNAc transferase appears to demonstrate promiscuity with regards to the hydroxyl-containing amino acid modified in short stretches of primary sequence of the glycosylated polypeptides.     

Characterization of intact N- and O-linked glycopeptides using higher energy collisional dissociation

Simultaneous elucidation of the glycan structure and the glycosylation site are needed to reveal the biological function of protein glycosylation. In this study, we employed a recent type of fragmentation termed higher energy collisional dissociation (HCD) to examine fragmentation patterns of intact glycopeptides generated from a mixture of standard glycosylated proteins. The normalized collisional energy (NCE) value for HCD was varied from 30 to 60% to evaluate the optimal conditions for the fragmentation of peptide backbones and glycoconjugates. Our results indicated that HCD with lower NCE values preferentially fragmented the sugar chains attached to the peptides to generate a ladder of neutral loss of monosaccharides, thereby enabling the putative glycan structure characterization. In addition, detection of the oxonium ions enabled unambiguous differentiation of glycopeptides from non-glycopeptides. In contrast, HCD with higher NCE values preferentially fragmented the peptide backbone and, thus, provided information needed for confident peptide identification. We evaluated the HCD approach with alternating NCE parameters for confident characterization of intact N- and O-linked glycopeptides in a single liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. In addition, we applied a novel data analysis pipeline, so-called GlycoFinder, to form a basis for automated data analysis. Overall, 38 unique intact glycopeptides corresponding to eight glycosylation sites (six N-linked and two O-linked sites) were confidently identified from a standard protein mixture. This approach provided concurrent characterization of both the peptide and the glycan, thereby enabling comprehensive structural characterization of glycoproteins in a single LC–MS/MS analysis.     

Enhanced peptide identification by electron transfer dissociation using an improved Mascot Percolator

Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%). We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.     

Negative-ion MALDI-MS2 for discrimination of α2,3- and α2,6-sialylation on glycopeptides labeled with a pyrene derivative

Here, we propose a novel method for the discrimination of α2,3- and α2,6-sialylation on glycopeptides. To stabilize the sialic acids, the carboxyl moiety on the sialic acid as well as the C-terminus and side chain of the peptide backbone were derivatized using 1-pyrenyldiazomethane (PDAM). The derivatization can be performed on the target plate for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), thereby avoiding complicated and time-consuming purification steps. After the on-plate PDAM derivatization, samples were subjected to negative-ion MALDI-MS using 3AQ-CHCA as a matrix. Deprotonated ions of the PDAM-derivatized form were detected as the predominant species without loss of sialic acid. The negative-ion collision-induced dissociation (CID) of PDAM-derivatized isomeric sialylglycopeptides, derived from hen egg yolk, showed characteristic spectral patterns. These data made it possible to discriminate α2,3- and α2,6-sialylation. In addition, sialyl isomers of a glycan with an asparagine could be discriminated based on their CID spectra. In brief, the negative-ion CID of PDAM-derivatized glycopeptides with α2,6-sialylation gave an abundant (0,2)A-type product ion, while that with α2,3-sialylation furnished a series of (2,4)A/Y-type product ions with loss of sialic acids. The unique fragmentation behavior appears to be derived from the difference of pyrene binding positions after ionization, depending on the type of sialylation. Thus, we show that on-plate PDAM derivatization followed by negative-ion MALDI-MS(2) is a simple and robust method for the discrimination of α2,3- and α2,6-sialylation on glycopeptides.     

Protein N-glycosylation is similar in the moss Physcomitrella patens and in higher plants

We have investigated the structure of glycans N-linked to the proteins of the moss Physcomitrella patens. The structural elucidation was carried out by western blotting using antibodies specific for N-glycan epitopes and by analysis of N-linked glycans enzymatically released from a total protein extract by combination of MALDI–TOF and MALDI–PSD mass spectrometry analysis. Nineteen N-linked oligosaccharides were characterised ranging from high-mannose-type and truncated paucimannosidic-type to complex-type N-glycans harbouring core-xylose, core-(1,3)-fucose and Lewis^a, as previously described for proteins from higher plants. This demonstrates that the processing of N-linked glycans, as well as the specificity of glycosidases and glycosyltransferases involved in this processing, are highly conserved between P. patens and higher plants. As a consequence, P. patens appears to be a new promising model organism for the investigation of the biological significance of protein N-glycosylation in the plant kingdom, taking advantage of the potential for gene targeting in this moss.Abbreviations Asn asparagine - CID collision-induced dissociation - Glc glucose - GlcNAc N-acetylglucosamine - Man mannose - MALDI–TOF MS matrix-assisted laser desorption ionization–time of flight mass spectrometry - PNGase A peptide N-glycosidase A - PSD post-source decay     

Electron transfer dissociation of iTRAQ labeled peptide ions

Triply and doubly charged iTRAQ ( isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD and supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/ z 162 yielded the reporter ion at m/ z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.     

Simultaneous Glycan-Peptide Characterization Using Hydrophilic Interaction Chromatography and Parallel Fragmentation by CID,Higher Energy Collisional Dissociation,and Electron Transfer Dissociation MS Applied to the N-Linked Glycoproteome of Campylobacter jejuni

Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or β-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.Campylobacter jejuni is a Gram-negative, microaerophilic, spiral-shaped, motile bacterium that is the most common cause of food- and water-borne diarrheal illness worldwide (1). Typical infections are acquired via the consumption of undercooked poultry where C. jejuni is found commensally (2). Symptoms in humans range from mild, non-inflammatory diarrhea to severe abdominal cramps, vomiting, and inflammation (3). Prior infection with C. jejuni is a common antecedent of two chronic immune-mediated disorders: Guillain-Barré syndrome (4) and immunoproliferative small intestine disease (5). A unique molecular trait of C. jejuni is the ability to post-translationally modify proteins by the N-linked addition of a 7-residue glycan (GalNAc-α1,4-GalNAc-α1,4-(Glcβ1,3)- GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-β1 where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucopyranose)) (6) at the consensus sequon (D/E)XNX(S/T) where X is any amino acid except proline (7).The N-linked C. jejuni heptasaccharide is encoded by the pgl (protein glycosylation) gene cluster (8–10), and the glycan is transferred to proteins by the PglB oligosaccharyltransferase (11) at the periplasmic face of the inner membrane (12). Removal of the N-glycosylation gene cluster (or indeed pglB alone) results in C. jejuni that displays poor adherence to and invasion of epithelial cell lines (13) and reduced colonization of the chicken gastrointestinal tract (14). Although this demonstrates a requirement for glycosylation in virulence, the proteins that mediate this are still unknown, and the overall role of glycan attachment remains to be elucidated. Our current understanding of the structural context of glycosylation in C. jejuni suggests that it does not play a role in steric stabilization by conferring structural rigidity as seen in eukaryotes (15) but occurs preferably on flexible loops and unordered regions of proteins (16–18). To investigate the role of glycosylation in protein function, recent studies have utilized mutagenesis to remove the N-linked sequon from three glycoproteins: Cj1496c (19), Cj0143c (20), and VirB10 (21). Removal of glycosylation from Cj1496c and Cj0143c had little effect on protein function; however, glycan attachment was required for correct localization of VirB10. Although the exact role of the glycan remains largely unknown, it appears to be site-specific with a single site, Asn⁹⁷, influencing localization of VirB10, whereas a second site, Asn³², is dispensable (21). It is clear that a more comprehensive analysis of the C. jejuni glycoproteome is required. A further complication in the elucidation of N-linked glycosylation is the use of the NCTC 11168 strain, which because of laboratory passage (22, 23) may not be the most appropriate model in which to study the virulence properties of glycan attachment. For example, we have recently shown that a surface-exposed virulence factor, JlpA, is glycosylated at two sites (Asn¹⁴⁶ and Asn¹⁰⁷) in all sequenced C. jejuni strains except NCTC 11168, which contains only Asn¹⁴⁶ (24).Glycoproteomics in C. jejuni is also a major technical challenge. Unlike eukaryotic N-linked glycans, the C. jejuni glycan is resistant to removal by protein N-glycosidase F (24) and chemical liberation via β-elimination (6) possibly because of the structure of the unique linking sugar, bacillosamine (25). Analysis therefore requires complementary methodology to elucidate the sites of glycosylation in the presence of the glycan. Preferential fragmentation of the glycan itself during collision-induced dissociation (CID) generally results in poor recovery of peptide fragment ions, and thus identification of the underlying protein and site of attachment remains problematic. MS³ has been attempted for site identification (6, 26); however, the data are limited by the requirement for sufficient ions for two rounds of tandem MS. We have also shown previously that C. jejuni encodes several hydrophobic integral membrane and outer membrane proteins possessing multiple transmembrane-spanning regions that are not amenable to gel-based approaches (27), particularly those using lectins for glycoprotein purification (28). We hypothesize that N-linked glycosylation is more widespread than previously demonstrated (6, 7, 26) because these studies examined only soluble proteins (6, 26) or used lectin affinity (6, 7), which limits the amount and type of detergents that can be used. Recent work (26) has demonstrated the potential of exploiting the hydrophilic nature of the C. jejuni glycan to enable glycopeptide enrichment.The ability to generate product ions useful for the identification of a glycosylated peptide is governed by three factors: the peptide backbone, the glycan, and the fragmentation approach. Multiple strategies exist to separately exploit the first two of these parameters (29, 30), but it is only recently that selective fragmentation of modified peptides has been available through electron transfer dissociation (ETD)¹ and electron capture dissociation (31, 32). ETD/electron capture dissociation enable the selective cleavage of the peptide while maintaining the carbohydrate structure, and this has been demonstrated using eukaryotic glycopeptides (33, 34) and more recently glycopeptides isolated from the pathogen Neisseria gonorrhoeae (35). A more recent fragmentation approach is higher energy collisional (C-trap) dissociation (HCD), which uses higher fragmentation energies than standard CID and enables identification of modifications, such as phosphotyrosine (36), via diagnostic immonium ions and high mass accuracy over the full mass range in MS/MS. HCD has not previously been applied to glycopeptides.We applied several enrichment and MS fragmentation approaches to the characterization of the glycoproteome of C. jejuni HB93-13. Sequence analysis determined that the HB93-13 genome contains 510 N-linked sequons ((D/E)XNX(S/T)) in 382 proteins of which 261 (with 371 potential N-linked sites) are predicted to pass through the inner membrane and are therefore the subset that may be glycosylated. We examined trypsin digests of whole cell and membrane protein preparations using zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and graphite enrichment of gel-separated proteins using several mass spectrometric techniques (CID, HCD, and ETD). This is the first study to demonstrate the potential of using the high energy fragmentation of HCD to overcome the signal disruption caused by labile glycan fragmentation and to provide peptide sequencing within a single step. Manual data analysis was also simplified as the GalNAc fragment ion (204.086 Da) provides a signature that can be used to highlight glycopeptides within a complex mixture. We identified 81 glycosylation sites, including 47 not described previously in the literature and a single site that cannot be unambiguously assigned. The majority of these are present on proteins not amenable to traditional gel-based analyses, such as hydrophobic transmembrane proteins. Our work more than doubles the previously known N-linked C. jejuni glycoproteome and provides a clear rationale for other studies where the peptide and glycan need to remain associated.     

Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei

Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.     

Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD

Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.     

Development of a combined chemical and enzymatic approach for the mass spectrometric identification and quantification of aberrant N-glycosylation

Direct mass spectrometric analysis of aberrant protein glycosylation is a challenge to the current analytical techniques. Except lectin affinity chromatography, no other glycosylation enrichment techniques are available for analysis of aberrant glycosylation. In this study, we developed a combined chemical and enzymatic strategy as an alternative for the mass spectrometric analysis of aberrant glycosylation. Sialylated glycopeptides were enriched with reverse glycoblotting, cleaved by endoglycosidase F3 and analyzed by mass spectrometry with both neutral loss triggered MS³ in collision induced dissociation (CID) and electron transfer dissociation (ETD). Interestingly, a great part of resulted glycopeptides were found with fucose attached to the N-acetylglucosamine (N-GlcNAc), which indicated that the aberrant glycosylation that is carrying both terminal sialylation and core fucosylation was identified. Totally, 69 aberrant N-glycosylation sites were identified in sera samples from hepatocellular carcinoma (HCC) patients. Following the identification, quantification of the level of this aberrant glycosylation was also carried out using stable isotope dimethyl labeling and pooled sera sample from liver cirrhosis and HCC was compared. Six glycosylation sites demonstrated elevated level of aberrancy, which demonstrated that our developed strategy was effective in both qualitative and quantitative studies of aberrant glycosylation.