Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii.Acinetobacter baumannii is an emerging opportunistic pathogen of increasing significance to health care institutions worldwide (1–3). The growing number of identified multiple drug resistant (MDR)¹ strains (2–4), the ability of isolates to rapidly acquire resistance (3, 4), and the propensity of this agent to survive harsh environmental conditions (5) account for the increasing number of outbreaks in intensive care, burn, or high dependence health care units since the 1970s (2–5). The burden on the global health care system of MDR A. baumannii is further exacerbated by standard infection control measures often being insufficient to quell the spread of A. baumannii to high risk individuals and generally failing to remove A. baumannii from health care institutions (5). Because of these concerns, there is an urgent need to identify strategies to control A. baumannii as well as understand the mechanisms that enable its persistence in health care environments.Surface glycans have been identified as key virulence factors related to persistence and virulence within the clinical setting (6–8). Acinetobacter surface carbohydrates were first identified and studied in A. venetianus strain RAG-1, leading to the identification of a gene locus required for synthesis and export of the surface carbohydrates (9, 10). These carbohydrate synthesis loci are variable yet ubiquitous in A. baumannii (11, 12). Comparison of 12 known capsule structures from A. baumannii with the sequences of their carbohydrate synthesis loci has provided strong evidence that these loci are responsible for capsule synthesis with as many as 77 distinct serotypes identified by molecular serotyping (11). Because of the non-template driven nature of glycan synthesis, the identification and characterization of the glycans themselves are required to confirm the true diversity. This diversity has widespread implications for Acinetobacter biology as the resulting carbohydrate structures are not solely used for capsule biosynthesis but can be incorporated and utilized by other ubiquitous systems, such as O-linked protein glycosylation (13, 14).Although originally thought to be restricted to species such as Campylobacter jejuni (15, 16) and Neisseria meningitidis (17), bacterial protein glycosylation is now recognized as a common phenomenon within numerous pathogens and commensal bacteria (18, 19). Unlike eukaryotic glycosylation where robust and high-throughput technologies now exist to enrich (20–22) and characterize both the glycan and peptide component of glycopeptides (23–25), the diversity (glycan composition and linkage) within bacterial glycosylation systems makes few technologies broadly applicable to all bacterial glycoproteins. Because of this challenge a deeper understanding of the glycan diversity and substrates of glycosylation has been largely unachievable for the majority of known bacterial glycosylation systems. The recent implementation of selective glycopeptide enrichment methods (26, 27) and the use of multiple fragmentation approaches (28, 29) has facilitated identification of an increasing number of glycosylation substrates independent of prior knowledge of the glycan structure (30–33). These developments have facilitated the undertaking of comparative glycosylation studies, revealing glycosylation is widespread in diverse genera and far more diverse then initially thought. For example, Nothaft et al. were able to show N-linked glycosylation was widespread in the Campylobacter genus and that two broad groupings of the N-glycans existed (34).During the initial characterization of A. baumannii O-linked glycosylation the use of selective enrichment of glycopeptides followed by mass spectrometry analysis with multiple fragmentation technologies was found to be an effective means to identify multiple glycosylated substrates in the strain ATCC 17978 (14). Interestingly in this strain, the glycan utilized for protein modification was identical to a single subunit of the capsule (13) and the loss of either protein glycosylation or glycan synthesis lead to decreases in biofilm formation and virulence (13, 14). Because of the diversity in the capsule carbohydrate synthesis loci and the ubiquitous distribution of the PglL O-oligosaccharyltransferase required for protein glycosylation, we hypothesized that the glycan variability might be also extended to O-linked glycosylation. This diversity, although common in surface carbohydrates such as the lipopolysaccharide of numerous Gram-negative pathogens (35), has only recently been observed within bacterial proteins glycosylation system that are typically conserved within species (36) and loosely across genus (34, 37).In this study, we explored the diversity within the O-linked protein glycosylation systems of Acinetobacter species. Our analysis complements the recent in silico studies of A. baumannii showing extensive glycan diversity exists in the carbohydrate synthesis loci (11, 12). Employing global strategies for the analysis of glycosylation, we experimentally demonstrate that the variation in O-glycan structure extends beyond the genetic diversity predicted by the carbohydrate loci alone and targets proteins of similar properties and identity. Using this knowledge, we developed a targeted approach for the detection of protein glycosylation, enabling streamlined analysis of glycosylation within a range of genetic backgrounds. We determined that; O-linked glycosylation is widespread in clinically relevant Acinetobacter species; inter- and intra-strain heterogeneity exist within glycan structures; glycan diversity, although extensive results in the generation of glycans with similar properties and that the utilization of a single glycan for capsule and O-linked glycosylation is a general feature of A. baumannii but may not be a general characteristic of all Acinetobacter species such as A. baylyi.     

The Prevalence and Nature of Glycan Alterations on Specific Proteins in Pancreatic Cancer Patients Revealed Using Antibody-Lectin Sandwich Arrays

Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value. However, an exploration of the population variation and prevalence of glycan alterations on specific proteins has been lacking because of limitations in conventional glycobiology methods. Here we report the use of a previously developed antibody-lectin sandwich array method to characterize both the protein and glycan levels of specific mucins and carcinoembryonic antigen-related proteins captured from the sera of pancreatic cancer patients (n = 23) and control subjects (n = 23). The MUC16 protein was frequently elevated in the cancer patients (65% of the patients) but showed no glycan alterations, whereas the MUC1 and MUC5AC proteins were less frequently elevated (30 and 35%, respectively) and showed highly prevalent (up to 65%) and distinct glycan alterations. The most frequent glycan elevations involved the Thomsen-Friedenreich antigen, fucose, and Lewis antigens. An unexpected increase in the exposure of α-linked mannose also was observed on MUC1 and MUC5ac, indicating possible N-glycan modifications. Because glycan alterations occurred independently from the protein levels, improved identification of the cancer samples was achieved using glycan measurements on specific proteins relative to using the core protein measurements. The most significant elevation was the cancer antigen 19-9 on MUC1, occurring in 19 of 23 (87%) of the cancer patients and one of 23 (4%) of the control subjects. This work gives insight into the prevalence and protein carriers of glycan alterations in pancreatic cancer and points to the potential of using glycan measurements on specific proteins for highly effective biomarkers.Alterations to the glycan structures on extracellular proteins are a common feature of many types of epithelial cancer such as pancreatic, colon, and breast cancers (1, 2). Cancer-associated glycan structures are thought to be functionally involved in many of the phenotypes characterizing cancer cells, including the ability to migrate, avoid apoptosis, evade immune destruction, and enter and exit the vasculature (3). Because proteins bearing cancer-associated glycans can be shed by tumor cells into the circulation, blood-based diagnostic tests using glycan detection may be possible. A potential advantage of using glycans for diagnostics is that carbohydrate modifications of particular proteins may be altered more frequently or more specifically in certain disease states than their underlying core protein concentrations. However, to evaluate and use such a strategy, the prevalence with which various structures appear and the specific proteins on which they appear must be better characterized.Previous studies of cancer-associated glycosylation using enzymatic, chromatographic, and mass spectrometry methods have been very effective for providing detailed information about the glycan structures produced by cancer cells, but because of the requirements for large amounts of material and the time involved to analyze each sample, these studies generally used either cell culture material or a small number of patient samples. Therefore, while many cancer-associated glycans have been identified, much remains unknown about these glycans, including how often they appear, how closely they are associated with particular disease states, and the distribution of protein carriers on which they appear.Affinity-based methods, using reagents such as lectins or glycan-binding antibodies, are a valuable complement to the above mentioned methods. Using antibodies or lectins that bind specific glycans, one may reproducibly measure the levels of those glycans over multiple samples. Although affinity-based glycosylation studies do not provide the structural detail provided by mass spectrometry and enzymatic methods, they can provide information about the biological variation of a particular motif.Lectins and glycan-binding antibodies have been used extensively in immunohistochemistry, for example in studies to examine the tissue distribution in pancreatic tumors of certain blood group carbohydrates (4, 5). Lectins have been valuable in immunoaffinity electrophoresis and blotting methods to identify cancer-associated glycan variants on major serum proteins such as α-fetoprotein (6), haptoglobin (7, 8), α₁-acid glycoprotein (9), and α₁-antitrypsin (10). Antibodies raised against particular glycan groups, such as the Thomsen-Friedenreich antigens (11), the Lewis blood group structures (12), and underglycosylated MUC1¹ (13) also have been used to study the roles of glycans in cancer. As a means of quantifying glycans on specific proteins, lectins have been used in the capture or detection of proteins in microtiter plates (14).We previously demonstrated an antibody-lectin sandwich array method (15) that is a valuable complement to the above methods and is ideal for profiling the prevalence of multiple glycans on multiple proteins. Glycan levels can be probed directly from biological samples, and many samples or detection conditions can be processed efficiently in a low volume, high throughput format (16). This method is complementary to lectin microarrays (17–19), which are useful for measuring glycan levels on individual, purified proteins; glycan microarrays (20, 21), which are used to measure the recognition of carbohydrate structures by various glycan-binding reagents; and glycoprotein arrays (22) for examining glycosylation on proteins isolated from biological samples.We applied this method to the study of glycan alterations on proteins in the circulation of pancreatic cancer patients. We sought to define the prevalence of various glycan alterations on particular protein carriers and to investigate whether those measurements have advantages for cancer diagnostics relative to measurements of core proteins. We designed antibody microarrays to target members of the mucin and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) families because some of those proteins are known to carry cancer-associated glycans. Mucins are extracellular, long-chain glycoproteins involved in the control and protection of epithelial surfaces, and the expression and glycosylation of several mucins are often altered and functionally involved in cancer (23, 24). The CEACAM family of proteins also is functionally involved in cancer, and they carry cancer-associated glycans (25, 26), but the glycans on CEACAMs are less well studied than those on mucins. By measuring both glycan levels and the core protein levels of several of these molecules, we were able to investigate whether alterations to glycans can appear at a higher rate than changes to core protein abundances. The ability to test the presence of glycan structures on multiple protein carriers in multiple samples was critical to investigating these questions.     

Structural and Quantitative Evidence for Dynamic Glycome Shift on Production of Induced Pluripotent Stem Cells

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2–3 to α-2–6, and the expression of α-1–2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a “glycome shift” upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs.Induced pluripotent stem cells (iPSCs)¹ are genetically manufactured pluripotent cells obtained by the transfection of reprogramming factors. Such iPSCs were first reported in 2006 for the mouse (1) and in 2007 for humans (2, 3). Although iPSCs have already been used in the fields of drug development and disease models (4–7), basic aspects of iPSCs largely remain to be elucidated to provide us with a fuller understanding of their properties and for therapeutic applications to be developed in the field of regenerative medicine. These aspects include the need for a definitive system to be established to evaluate their properties; e.g. pluripotency, differentiation propensity, risk of possible contamination of xenoantigens, and even the potential for tumorigenesis. Cell surface glycans are often referred to as the “cell signature,” which changes dramatically depending on the cell properties and conditions (8) as a result of changes in gene expression, including epigenetic modifications of glycan-related molecules. Glycans, because of their outermost cell-surface locations and structural complexity, are considered to be most advantageous communication molecules, playing roles in various biological phenomena. Indeed, SSEA3/4 and Tra-1–60/81, which have been used to discriminate pluripotency, are cell surface glycan epitopes that respond to some specific antibodies (9–12).Glycan-mediated cell-to-cell interactions have been shown to play important roles in various biological phenomena including embryogenesis and carcinogenesis (13–16). This might also be the case for the acquisition and maintenance of iPSC and ESC pluripotency, although there remains much to clarify concerning the roles of cell surface glycans in these events. Thus, the development of novel cell surface markers to evaluate the properties of iPSCs and ESCs is keenly required. Toward this goal, a glycomic approach has been made by several groups (17–20). In our previous study using an advanced lectin microarray technique (21), thirty-eight lectins capable of discriminating between iPSCs and SCs were statistically selected, and the characteristic features of the pluripotent state were obtained. The glycan profiles of the parent SCs, derived from four different tissues, were totally different from one another and from those of the iPSCs. Despite this observation, the technique used lacks the ability to determine detailed glycan structures or allow their quantification. For this purpose, a conventional approach based on high performance liquid chromatography (HPLC) combined with matrix-assisted laser desorption-ionization (MALDI) - time of flight (TOF) mass spectrometry (MS) was undertaken for both the definitive identification of glycan structures and their quantitative comparison, which remained unclear in the previous analysis (21).We report here structural data on N-linked and O-linked glycans derived from the human iPSC 201B7 cell line (2) and human dermal fibroblasts (SC) representing undifferentiated and differentiated cells, respectively. For quantitative comparison, the glycans were liberated by gas-phase hydrazinolysis from similar numbers of cells (22–25) fluorescently tagged with 2-aminopyridine (2-AP) at their reducing terminus (26, 27), following which the derived pyridylaminated (PA-) glycans were purified by multiple-mode (i.e. anion-exchange, size-fractionation and reverse-phase) HPLC. Their structures were determined and quantified by HPLC mapping assisted with MALDI-TOF-MS and exoglycosidase digestion analyses. This report thus provides the first structural evidence showing the occurrence of a dynamic “glycome shift” upon induction of pluripotency.     

Comparative Performance of Four Methods for High-throughput Glycosylation Analysis of Immunoglobulin G in Genetic and Epidemiological Research

The biological and clinical relevance of glycosylation is becoming increasingly recognized, leading to a growing interest in large-scale clinical and population-based studies. In the past few years, several methods for high-throughput analysis of glycans have been developed, but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. In this study, we compared liquid chromatography, capillary gel electrophoresis, and two MS methods for quantitative profiling of N-glycosylation of IgG in the same data set of 1201 individuals. To evaluate the accuracy of the four methods we then performed analysis of association with genetic polymorphisms and age. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and multiplexed capillary gel electrophoresis, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should inform the selection of the most appropriate method in future studies.Glycans are important structural and functional components of the majority of proteins, but because of their structural complexity and the absence of a direct genetic template our current understanding of the role of glycans in biological processes lags significantly behind the knowledge about proteins or DNA (1, 2). However, a recent comprehensive report endorsed by the US National Academies concluded that “glycans are directly involved in the pathophysiology of every major disease and that additional knowledge from glycoscience will be needed to realize the goals of personalized medicine” (3).It is estimated that the glycome (defined as the complete set of all glycans) of a eukaryotic cell is composed of more than a million different glycosylated structures (1), which contain up to 10,000 structural glycan epitopes for interaction with antibodies, lectins, receptors, toxins, microbial adhesins, or enzymes (4). Our recent population-based studies indicated that the composition of the human plasma N-glycome varies significantly between individuals (5, 6). Because glycans have important structural and regulatory functions on numerous glycoproteins (7), the observed variability suggests that differences in glycosylation might contribute to a large part of the human phenotypic variability. Interestingly, when the N-glycome of isolated immunoglobulin G (IgG)¹ was analyzed, it was found to be even more variable than the total plasma N-glycome (8), indicating that the combined analysis of all plasma glycans released from many different glycoproteins blurs signals of protein-specific regulation of glycosylation.A number of studies have investigated the role of glycans in human disease, including autoimmune diseases and cancer (9, 10). However, most human glycan studies have been conducted with very small sample sizes. Given the complex causal pathways involved in pathophysiology of common complex disease, and thus the likely modest effect sizes associated with individual factors, the majority of these studies are very likely to be substantially underpowered. In the case of inflammatory bowel disease, only 20% of reported inflammatory bowel disease glycan associations were replicated in subsequent studies, suggesting that most are false positive findings and that there is publication bias favoring the publication of positive findings (11). This situation is similar to that which occurred in the field of genetic epidemiology in the past when many underpowered candidate gene studies were published and were later found to consist of mainly false positive findings (12, 13). It is essential, therefore, that robust and affordable methods for high-throughput analysis are developed so that adequately powered studies can be conducted and the publication of large numbers of small studies reporting false positive results (which could threaten the credibility of glycoscience) be avoided.Rapid advances of technologies for high-throughput genome analysis in the past decade enabled large-scale genome-wide association studies (GWAS). GWAS has become a reliable tool for identification of associations between genetic polymorphisms and various human diseases and traits (14). Thousands of GWAS have been conducted in recent years, but these have not included the study of glycan traits until recently. The main reason was the absence of reliable tools for high-throughput quantitative analysis of glycans that could match the measurements of genomic, biochemical, and other traits in their cost, precision, and reproducibility. However, several promising high-throughput technologies for analysis of N-glycans were developed (8, 15–20) recently. Successful implementation of high-throughput analytical techniques for glycan analysis resulted in publication of four initial GWAS of the human glycome (21–24).In this study, we compared ultra-performance liquid chromatography with fluorescence detection (UPLC-FLR), multiplex capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography electrospray mass spectrometry (LC-ESI-MS) as tools for mid-to-high-throughput glycomics and glycoproteomics. We have analyzed IgG N-glycans by all four methods in 1201 individuals from European populations. The analysis of associations between glycans and ∼300,000 single-nucleotide genetic polymorphisms was performed and correlation between glycans and age was studied in all four data sets to identify the analytical method that shows the strongest potential to uncover biological mechanisms underlying protein glycosylation.     

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics.Protein glycosylation is a biologically significant and complex post-translational modification, involved in cell-cell and receptor-ligand interactions (1–4). In fact, clinical biomarkers and therapeutic targets are often glycoproteins (5–9). Comprehensive glycoprotein characterization, involving glycosylation site identification, glycan structure determination, site occupancy, and glycan isoform distribution, is a technical challenge particularly for quantitative profiling of complex protein mixtures (10–13). Both N- and O-glycans are structurally heterogeneous (i.e. a single site may have different glycans attached or be only partially occupied). Therefore, the MS¹ signals from glycopeptides originating from a glycoprotein are often weaker than from non-glycopeptides. In addition, the ionization efficiency of glycopeptides is low compared with that of non-glycopeptides and is often suppressed in the presence of non-glycopeptides (11–13). When the MS signals of glycopeptides are relatively high in simple protein digests then diagnostic sugar oxonium ion fragments produced by, for example, front-end collisional activation can be used to detect them. However, when peptides and glycopeptides co-elute, parent ion scanning is required to selectively detect the glycopeptides (14). This can be problematic in terms of sensitivity, especially for detecting glycopeptides in digests of complex protein extracts.Isolation of glycopeptides from proteolytic digests of complex protein mixtures can greatly enhance the MS signals of glycopeptides using reversed-phase LC-ESI-MS (RPLC-ESI-MS) or MALDI-MS (15–24). Hydrazide chemistry is used to isolate, identify, and quantify N-linked glycopeptides effectively, but this method involves lengthy chemical procedures and does not preserve the glycan moieties thereby losing valuable information on glycan structure and site occupancy (15–17). Capturing glycopeptides with lectins has been widely used, but restricted specificities and unspecific binding are major drawbacks of this method (18–21). Under reversed-phase LC conditions, glycopeptides from tryptic digests of gel-separated glycoproteins have been enriched using graphite powder medium (22). In this case, however, a second digestion with proteinase K is required for trimming down the peptide moieties of tryptic glycopeptides so that the glycopeptides (typically <5 amino acid residues) essentially resemble the glycans with respect to hydrophilicity for subsequent separation. Moreover, the short peptide sequences of the proteinase K digest are often inadequate for de novo sequencing of the glycopeptides.Glycopeptide enrichment under normal-phase LC (NPLC) conditions has been demonstrated using various hydrophilic media and different capture and elution conditions (23–28). NPLC allows either direct enrichment of peptides modified by various N-linked glycan structures using a ZIC®-HILIC column (23–27) or targeting sialylated glycopeptides using a titanium dioxide micro-column (28). However, NPLC is neither effective for enriching less hydrophilic glycopeptides, e.g. the five high mannose type glycopeptides modified by 7–11 monosaccharide units from a tryptic digest of ribonuclease b (RNase B), nor for enriching O-linked glycopeptides of bovine fetuin using a ZIC-HILIC column (23). The use of Sepharose medium for enriching glycopeptides yielded only modest recovery of glycopeptides (28). In addition, binding of hydrophilic non-glycopeptides with these hydrophilic media contaminates the enriched glycopeptides (23, 28).We have recently developed an ion-pairing normal-phase LC (IP-NPLC) method to enrich glycopeptides from complex tryptic digests using Sepharose medium and salts or bases as ion-pairing reagents (29). Though reasonably effective the technique still left room for significant improvement. For example, the method demonstrated relatively modest glycopeptide selectivity, providing only 16% recovery for high mannose type glycopeptides (29). Here we report on a new IP-NPLC method using acids as ion-pairing reagents and polyhydroxyethyl aspartamide (A) as the stationary phase for the effective isolation of tryptic glycopeptides. The method was developed and evaluated using a tryptic digest of RNase B and fetuin mixture. In addition, we demonstrate that O-linked glycopeptides can be effectively isolated from a fetuin tryptic digest by IP-NPLC after removal of the N-linked glycans by PNGase F.The new IP-NPLC method was used to enrich N-linked glycopeptides from the tryptic digests of protein extracts of wild-type (wt) and PglD mutant strains of Campylobacter jejuni NCTC 11168. C. jejuni has a unique N-glycosylation system that glycosylates periplasmic and inner membrane proteins containing the extended N-linked sequon, D/E-X-N-X-S/T, where X is any amino acid other than proline (30–32). The N-linked glycan of C. jejuni has been previously determined to be GalNAc-α1,4-GalNAc-α1,4-[Glcβ1,3]-GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-β1 (BacGalNAc₅Glc residue mass: 1406 Da), where Bac is 2,4-diacetamido-2,4,6-trideoxyglucopyranose (30). In addition, the glycan structure of C. jejuni is conserved, unlike in eukaryotic systems (30–32). IP-NPLC recovered close to 100% of the bacterial N-linked glycopeptides with virtually no contamination of non-glycopeptides. Furthermore, we demonstrate for the first time that acetylation of bacillosamine is incomplete in the wt using IP-NPLC and label-free MS.     

N-glycomic Profiling as a Tool to Separate Rectal Adenomas from Carcinomas

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world''s three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I–II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.Glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans, that cover all human cells. Around 1% of the human genome participates in the biosynthesis of glycans(1). This biosynthesis is the most complex post-translational modification of proteins, and the great variability in glycan structures contains a tremendous ability to fine-tune the chemical and biological properties of glycoproteins. The glycosylation process occurs most abundantly in the Golgi apparatus and the endoplasmic reticulum, but also occurs in the cytoplasm and nucleus (2). Most glycoconjugates are localized to cell surfaces, where glycans participate in events essential for cell viability and function, such as cell adhesion, motility, and intracellular signaling (2). Changes in these functions are key steps seen when normal cells transform to malignant ones, and these are also reflected in changes of a cell''s glycan profile, observed in many cancers (3, 4). Specific structural changes in glycans may serve as cancer biomarkers (5, 6), and changes in glycosylation profiles are related to aggressive behavior in tumor cells (7–9).Cancer-associated asparagine-linked glycan (N-glycan) structures may play specific roles in supporting tumor progression; growth (10, 11), invasion (12, 13), and angiogenesis (14). Changes in the N-glycan profile emerge in numerous cancers, including lung (15, 16), breast (17), and colorectal cancer (CRC)¹ (16, 18). Balog et al. (18) comparing the N-glycomic profile of CRC tissue to adjacent normal mucosa, reported differences in specific glycan structures. Moreover, serum N-glycosylation profile from patients with CRC differ from those of healthy controls (19).Colorectal cancer is the third most common cause of cancer-related death worldwide and its incidence is rising; 40% of CRCs are of rectal origin. Roughly 40% of patients have localized disease (stage I–II; Dukes A–B), another 40% loco regional disease (stage III; Dukes C), and 20% metastasized disease (stage IV; Dukes D) (20). Although stage at diagnosis is the most important factor determining prognosis, clinical outcome, and response to adjuvant treatment can markedly vary within each stage. Adjuvant therapy routinely goes to stage III patients, but the benefit of adjuvant treatment for stage II patients is unclear. Of stage II patients, 80% are cured by radical surgery alone. To identify patients who will benefit from postoperative treatment, we need novel biomarkers. The glycan profile of the tumor tissue could provide new biomarkers for diagnosis and prognosis of cancer.In this study, we characterized the N-glycomic profiles of rectal adenomas and carcinomas by MALDI-TOF mass spectrometric (MS) profiling of asparagine-linked glycans. Our aim was to identify differences between adenomas and carcinomas, and also between cancers of different stages. Based on glycan profiling, we also chose, for immunohistochemical expression studies of a series of 220 CRC patients, two glycan markers: sialyl Lewis a and pauci-mannose.     

SweetSEQer,Simple de Novo Filtering and Annotation of Glycoconjugate Mass Spectra

The past 15 years have seen significant progress in LC-MS/MS peptide sequencing, including the advent of successful de novo and database search methods; however, analysis of glycopeptide and, more generally, glycoconjugate spectra remains a much more open problem, and much annotation is still performed manually. This is partly because glycans, unlike peptides, need not be linear chains and are instead described by trees. In this study, we introduce SweetSEQer, an extremely simple open source tool for identifying potential glycopeptide MS/MS spectra. We evaluate SweetSEQer on manually curated glycoconjugate spectra and on negative controls, and we demonstrate high quality filtering that can be easily improved for specific applications. We also demonstrate a high overlap between peaks annotated by experts and peaks annotated by SweetSEQer, as well as demonstrate inferred glycan graphs consistent with canonical glycan tree motifs. This study presents a novel tool for annotating spectra and producing glycan graphs from LC-MS/MS spectra. The tool is evaluated and shown to perform similarly to an expert on manually curated data.Protein glycosylation is a common modification, affecting ∼50% of all expressed proteins (1). Glycosylation affects critical biological functions, including cell-cell recognition, circulating half-life, substrate binding, immunogenicity, and others (2). Regrettably, determining the exact role glycosylation plays in different biological contexts is slowed by a dearth of analytical methods and of appropriate software. Such software is crucial for performing and aiding experts in data analysis complex glycosylation.Glycopeptides are highly heterogeneous in regard to glycan composition, glycan structure, and linkage stereochemistry in addition to the tens of thousands of possible peptides. The analysis of protein glycosylation is often segmented into three distinct types of mass spectrometry experiments, which together help to resolve this complexity. The first analyzes enzymatically or chemically released glycans (which may or may not be chemically modified), and the second determines glycosylation sites after release of glycans from peptides (the resulting mass spectra allow detection of glycosylation sites and the glycans on those sites simultaneously). The third determines the glycosylation sites and the glycans on those sites simultaneously, by MS of intact glycopeptides. Frequently, researchers will perform all three types of analysis, with the first two types providing information about possible combinations of glycan structures and peptides that could be found in the third experiment. Using this MS1 information, the problem is reduced to matching masses observed with a combinatorial pool of all possible glycans and all possible glycosylated peptides within a sample; however, this combinatorial approach alone is insufficient (3), and tandem mass spectrometry can provide copious additional information to help resolve the glycopeptide content from complex samples.The similar problem of inferring peptide sequences from MS/MS spectra has received considerably more attention. Peptide inference is more constrained than glycan inference, because the chain of MS/MS peaks corresponds to a linear peptide sequence; given an MS/MS spectrum, the linear peptide sequence can be inferred through brute force or dynamic programming via de novo methods (4–6) as described in Ref. 7. Additionally, the possible search space of peptides can be dramatically lowered by using database searching (8–21) as described in Ref. 7, which compares the MS/MS spectrum to the predicted spectra from only those peptides resulting from a protein database or translated open reading frames (ORFs) of a genomic database.The possible search space of glycans is larger than the search space of peptides because, in contrast to linear peptide chains, glycans may form branching trees. Identifying glycans using database search methodologies is impractical, as it is impractical to define the database when the detailed activities of the set of glycosyltransferases are not defined. Generating an overly large database would artificially inflate the set of incompletely characterized spectra, and too small of a search space would lead to inaccurate results. Furthermore, as glycosylation is not a template-driven process, no clear choice for a database matching approach is available, and de novo sequencing is therefore a more appropriate approach.As a result, few desirable software options are available for the high throughput analysis of tandem mass spectrometry data from intact glycopeptides (as noted in a recent review (22)). In fact, manual annotation of spectra is still commonplace, despite being slow and despite the potential for disagreement between different experts. Some available software requires user-defined lists of glycan and/or peptide masses as input, which is suboptimal from a sample consumption and throughput perspective (23, 24). These lists must typically be generated by parallel experiments or simply hypothesized a priori, meaning omissions in either list may affect the results. Furthermore, some software does not work on batched input files, meaning each spectrum must be analyzed separately (23, 25–28). Moreover, there is an even greater lack of open source software for glycoproteomics, so modifying the existing software for the researchers individual applications is not easily achieved. The one open source tool that we know of (GlypID) is applicable only to the analysis of glycopeptide spectra acquired from a very specialized workflow, which requires MS1, CID, and higher-energy C-trap type dissociation (HCD) spectra (29). With that approach, oxonium ions from HCD spectra are necessary to predict the glycan class; potential peptide lists are queried by precursor m/z values (requiring accurate a priori knowledge of all modifications), and possible theoretical “N-linked” precursor m/z values are used to select candidate spectra (using templates, unlike de novo characterization). As a result, the tool is specialized and limited to analysis of “N-linked” glycopeptide spectra from very specific experimental setups.Free, open-source glycoproteomic software capable of batch analysis of general tandem mass spectrometry spectra of glycoconjugates is sorely needed. In this work, we present SweetSEQer, a tool for de novo analysis of tandem mass spectra of glycoconjugates (the most general class of spectra containing fragmentation involving sugars). Furthermore, because SweetSEQer is so general and simple, and because it does not require specific experimental setup, it is widely applicable to the analysis of general glycoconjugate spectra (e.g. it is already applicable to “O-linked” glycopeptide and glycoconjugate spectra). Moreover, because it is an open source and does not use external software, it not only eschews solving problems like MS1 deisotoping, it can also be easily customized and even used to augment and complement existing tools like GlypID (and, because we do not use a “copyleft” software license, our algorithm and code can even be added to non-open source and proprietary variants).SweetSEQer''s performance was tested on a validated, manually annotated set of glycoconjugate identifications from a urinary glycoproteomics study. Specificity was demonstrated by showing a low identification rate on negative control spectra from Escherichia coli. Annotated structures are shown to be consistent by a human expert by demonstrating a high overlap in identified glycan fragment ions, as well as a consistency between SweetSEQer''s predicted glycan graph and glycan chains produced by an expert. Our simple object-oriented python implementation is freely available (Apache 2.0 license) on line.     

In Situ trans Ligands of CD22 Identified by Glycan-Protein Photocross-linking-enabled Proteomics

CD22, a regulator of B-cell signaling, is a siglec that recognizes the sequence NeuAcα2–6Gal on glycoprotein glycans as ligands. CD22 interactions with glycoproteins on the same cell (in cis) and apposing cells (in trans) modulate its activity in B-cell receptor signaling. Although CD22 predominantly recognizes neighboring CD22 molecules as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics scale study to identify candidate trans ligands of CD22 on B-cells by UV photocross-linking CD22-Fc chimera bound to B-cell glycoproteins engineered to carry sialic acids with a 9-aryl azide moiety. Using mass spectrometry-based quantitative proteomics to analyze the cross-linked products, 27 glycoproteins were identified as candidate trans ligands. Next, CD22 expressed on the surface of one cell was photocross-linked to glycoproteins on apposing B-cells followed by immunochemical analysis of the products with antibodies to the candidate ligands. Of the many candidate ligands, only the B-cell receptor IgM was found to be a major in situ trans ligand of CD22 that is selectively redistributed to the site of cell contact upon interaction with CD22 on the apposing cell.Glycan-binding proteins (GBPs)¹ mediate diverse aspects of cell communication through their interactions with their counter-receptors comprising glycan ligands carried on cell surface glycoproteins and glycolipids. Identification of the in situ counter-receptors of glycan-binding proteins is problematic due to the fact that the vast majority of the glycoproteins of a cell will carry highly related glycan structures because they share the same secretory pathway that elaborates their glycans post-translationally en route to the cell surface. Thus, although many glycoproteins will carry the glycan structure recognized by a GBP, the challenge is to determine whether one, several, or all of these cell surface glycoproteins (and glycolipids) are recognized in situ as physiologically relevant counter-receptors (1–4). Standard in vitro methods, such as co-precipitation from cell lysates or Western blotting using binding protein probes, are useful for identifying glycoproteins that contain the glycan structure recognized by the GBP. However, these may not be relevant ligands in situ due to constraints imposed by their microdomain localization and the geometric arrangement of their glycans relative to the GBP presented on the apposing cell.In this report, we examine the in situ ligands of CD22 (Siglec-2), a member of the siglec family and a regulator of B-cell receptor (BCR) signaling that recognizes glycans containing the sequence NeuAcα2–6Gal as ligands (2, 5, 6). Regulation of BCR signaling by CD22 is effected by its proximity to the BCR through recruitment of a tyrosine phosphatase, SHP-1, which is in turn influenced by CD22 binding to its glycan ligands (6). Glycoproteins bearing CD22 ligands are abundantly expressed on B-cells and bind to CD22 in cis (on the same cell) (7), regulating BCR signaling (2, 5, 6). Although binding to cis ligands has been shown to “mask” CD22 from binding low avidity synthetic sialoside probes (2, 7), CD22 can also interact with ligands on apposing immune cells in trans (8–10). Interactions of CD22 with trans ligands influence T-cell signaling in vitro (11, 12), mediate B-cell homing via binding to sinusoidal endothelial cells in the bone marrow (13), and aid in “self”-recognition (14). Thus, interactions with both cis and trans ligands modulate CD22 function in immune homeostasis.Several groups have demonstrated that recombinant CD22-Fc chimera is capable of binding and precipitating the majority of glycoproteins from B- and T-cell lysates whose glycans contain the sequence NeuAcα2–6Gal (15–18). Among them, CD45, IgM, and CD22 itself were identified as specific B-cell binding partners and were postulated to have functional significance as in situ cis ligands of CD22 in regulation of BCR signaling (11, 16, 18–20). Several reports have also documented in situ interactions of CD22 with IgM and CD45, but these interactions were found to be of low stoichiometry and sialic acid-independent (19–21), leaving open the question of which glycoproteins served as in situ cis ligands of CD22 on B-cells that masked the glycan ligand binding site of CD22 (7). Subsequently, using metabolically labeled B-cells with sialic acids containing a photoactivatable 9-aryl azide moiety, we demonstrated that CD22 could be photocross-linked to its cis ligands, effectively tagging the in situ cis ligands with CD22 (15). Notably, there was no cross-linking observed to IgM or CD45, demonstrating that they are not significant in situ cis ligands of CD22 (15). Instead, only glycans of neighboring CD22 molecules interacted significantly with CD22, resulting in photocross-linking of homomultimeric complexes of CD22. Thus, despite the fact that most B-cell glycoproteins are recognized in vitro, CD22 selectively recognizes glycans of neighboring CD22 molecules as cis ligands in situ.With the perspective gained from analysis of cis ligands, we wished to determine whether CD22 was also selective in recognition of trans ligands upon cell contact. We have previously demonstrated that CD22 is redistributed to sites of cell contact of interacting B-cells and T-cells and that redistribution is mediated by the interaction of CD22 with sialic acid-containing trans ligands on the apposing cell (8). Stamenkovic et al. (22) had previously demonstrated that binding of T-cells to CD22-expressing COS cells was blocked by an anti-CD45RO antibody, suggesting that CD45 was a functional trans ligand of CD22 on T-cells. However, we found that redistribution of CD22 to sites of cell contact was also observed with CD45-deficient B-cells (8), indicating that, at a minimum, other glycoproteins must also serve as trans ligands of CD22 on B-cells.To assess whether CD22 recognizes all or a subset of glycoproteins as trans ligands on an apposing cell, we initiated an unbiased analysis of the trans ligands of CD22 on apposing B-cells using our protein-glycan cross-linking strategy (15). By cross-linking CD22-Fc to intact B-cells, we identified 27 candidate trans ligands of CD22 by quantitative mass spectrometry-based proteomics. We then looked at the in situ trans interactions of CD22 in the physiologically relevant cellular context by cross-linking CD22 expressed on one cell to the trans ligands with photoreactive sialic acids on the apposing cell. Our results indicate that only a subset of cell surface glycoproteins, including IgM and, to a lesser extent, CD45 and Basigin, are selectively recognized in trans by CD22. Indeed, IgM in particular is a preferred trans ligand that is selectively redistributed to the sites of cell contact on apposing B-cells in a CD22- and sialic acid-dependent manner despite a vast excess of cell surface glycoproteins that carry a glycan recognized by CD22. The results support the view that factors other than glycan sequence are critical for the in situ engagement of glycan-binding proteins with glycan ligand bearing counter-receptors on the same cell (in cis) or apposing cell (in trans).     

Glycomic Analysis of Life Stages of the Human Parasite Schistosoma mansoni Reveals Developmental Expression Profiles of Functional and Antigenic Glycan Motifs

Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galβ1–4(Fucα1–3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcβ1–4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galβ1–3(Galβ1–6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome''s glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.Schistosoma blood flukes give rise to infections in over 200 million people in developing countries worldwide (1). With a Disability-Adjusted Life Years (DALY) value of more than 3 million, schistosomiasis ranks as one of the neglected tropical diseases with the highest impact on public health (2). The schistosome has a complex and intriguing lifecycle, which involves a definitive host (mammal) as well as an intermediate host (snail). Infections with Schistosoma mansoni, one of the major schistosome species infecting humans, are initiated when snail-borne cercariae penetrate intact skin. The cercariae then transform into schistosomula, which enter the vasculature of the host and mature while migrating to the portal system. Here, adult male and female worms pair, with the female worm producing hundreds of eggs each day during a life span of several years unless the infection is treated by chemotherapy. Miracidia develop inside the maturing eggs while they cross the intestinal wall over a period of several days to be excreted with the feces. Miracidia then hatch from the eggs upon contact with fresh water and infect the snail host where asexual replication takes place and eventually new cercariae are shed. Notably, many eggs get trapped in organs such as the liver, where they induce a granulomatous inflammation and organ damage, the main cause of pathology in schistosomiasis (1).Throughout their lifecycle, schistosomes express a multitude of protein- and lipid-linked glycans that play an important role in the parasite biology. The expression of many glycan elements appears to be developmentally regulated by the differential expression of glycosyltransferases during the different lifecycle stages (3). A series of papers has been published indicating that schistosome glycans play essential roles in the molecular interaction of the parasite and the host immune system, enabling survival of the parasite and allowing chronic infection to establish. For example, glycosylated soluble egg antigens (SEA) interact with the C-type lectins mannose receptor (MR), macrophage galactose-type lectin (MGL) and dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and some of these interactions lead to immunomodulatory effects of specific components of SEA via dendritic cells (DCs)¹ (4, 5). Furthermore, fucosylated egg glycolipids trigger innate immune responses of peripheral blood mononuclear cells and egg glycans are required for periovular granuloma formation in a mouse model. In addition, cercarial secretions induce alternatively activated macrophages in a carbohydrate dependent manner (6–9). Importantly, also adaptive immune responses to schistosome glycans are mounted by the human host. A large part of the antibody responses to schistosomes is directed against antigenic glycan motifs, raising the question whether they could form a basis for antischistosome vaccine strategies (10).Rapid developments in mass spectrometry-based glycan-analysis technology in the last two decades have led to several studies focused on elucidating the glycan structures of somatic and secretory schistosome preparations (11–22). Among the typical glycan elements detected in S. mansoni were unusual and antigenic Fucα1–2Fucα1–3- (DF-) motifs attached to GalNAcβ1–4GlcNAc (LacDiNAc or LDN) (12, 14, 17–19, 21), Xylβ1–2- and Fucα1–3-modified N-glycan core structures (13, 15, 17, 20), and a unique O-glycan core (Galβ1–3(Galβ1–6)GalNAc) (14, 17) (see supplemental Table S5 for a definition of glycan motifs of S. mansoni glycoconjugates). Also more widely occurring glycan elements shared with the mammalian or snail host were detected, e.g. Galβ1–4GlcNAc (LacNAc or LN), Galβ1–4(Fucα1–3)GlcNAc (Lewis X or LeX), LDN, and GalNAcβ1–4(Fucα1–3)GlcNAc (LDN-F) (23, 24). These data were generated over a long period of time, often focusing on a single schistosome life stage and a specific class of glycans only, and using various analytical techniques and strategies that make inter-study comparisons often difficult. In addition, glycosylation of the schistosomula that develop shortly after infection and are considered to be relatively vulnerable to immune attack, has remained largely unexplored (20, 25, 26), although these could be interesting therapeutic targets (27–29). Clearly, an integrated and complete overview of schistosome glycosylation was so far not available.In this study, we therefore set out to determine the overall schistosome protein- and lipid-linked glycome by analyzing a total of 16 lifecycle stages ranging from cercariae to miracidia. We analyzed the glycoprotein-derived N- and O-glycans as well as the lipid-derived glycans of these life stages by a MALDI-TOF MS-based approach complemented with fragmentation and enzyme degradation studies. Our findings give new insights in the glycobiology of parasite development and parasite–host interaction and contribute to the identification of new potential immune intervention targets.     

Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications

Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited.Here, we present a detailed PTM¹ characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.Allergic respiratory disease is a global health problem and current clinical guidelines recommend a combination of allergen avoidance, pharmacotherapy, and allergen specific immunotherapy for treatment (1–4). At present allergy testing and vaccines are based on isolated crude antigen preparations from natural sources (i.e. HDM, pollens, etc.), but a move toward recombinant allergen design is ongoing (5, 6). This could have important functional implications because the production host will determine the repertoire of post-translational modifications (PTMs) and in particular glycan modifications presented on allergens.The carbohydrate structures found on allergens are in most cases not found in mammals and therefore frequently lead to the induction IgE antibodies named Cross-reactive Carbohydrate Determinants (CCD) (7–11). Moreover, glycans may directly be involved in and promote uptake and target allergens to carbohydrate lectin receptors on antigen presenting cells (APC) (12–14). Therefore, a full structural characterization of the glycans on the natural allergens is a prerequisite for understanding both antibody reactivity and lectin receptor mediated allergen recognition and modulation of the immune response (15, 16). Furthermore, a detailed characterization of PTMs of allergens is important for standardization of allergen products for diagnostic purposes as well as for vaccine use (17, 18). Although many major allergens and their etiology have been characterized in some detail, structural information on for example their immunological important PTM status is still incomplete (19–21).Mass spectrometry-based technologies offer sensitive and accurate analyses for identification and characterization of proteins. The common proteomics workflow typically adopts the bottom-up approach, i.e. in vitro proteolytic digestion of proteins followed by nanoflow-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) for protein identification and PTM characterization. Electron- or collision-driven fragmentation techniques, e.g. electron transfer dissociation (ETD) (22) or higher energy collisional dissociation (HCD) (23) have enabled accurate identification of peptides of purified proteins, e.g. allergens (21, 24), or complex biological samples (25–27) with concurrent characterization of their PTMs. One advantage of bottom-up mass spectrometry is the ability to resolve modified peptides within a narrow chromatographic time frame thereby enabling in-depth characterization of site-specific features, e.g. glycoforms, on peptides. This peptide-level information is subsequently used to generate a protein-level view on the PTM status for a given protein. Importantly, the PTM connectivity of the protein (28) is lost upon proteolytic digestion, and alternative approaches are often required for comprehensive characterization of all proteoforms (29). Top-down mass spectrometry has emerged as an alternative approach to bottom-up proteomics, offering complementary MS and MS/MS information that may be used for protein identification and characterization (30, 31). With top-down MS, intact proteins are typically analyzed by high-resolution FTMS and characterized at the MS/MS level by CID, HCD, ECD, or ETD. This technique provides instant protein-level information on analytes, e.g. sequence variants, amino acid substitutions, PTMs, etc., which can be verified at the MS/MS level by different fragmentation modes. The combination of bottom-up and top-down mass spectrometry is therefore a powerful tool for the identification and characterization of proteins. Here, we combine top-down and bottom-up mass spectrometry for comprehensive characterization of seven major allergens as a first step toward unraveling the molecular mode of action of allergens with complex PTMs. By these methods, we demonstrate hitherto unknown PTMs of HDM allergens and identify more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens. The new findings implicate important roles for carbohydrates in allergen recognition and response by the immune system.     

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.Immunoglobulins are key players of the human immune system. Immunoglobulin G (IgG)¹ is the most abundant representative of this group, with serum concentrations of ∼10 mg/ml (1). It consists of two heavy chains (γ-chains) made up of three constant regions (C_H1, C_H2, and C_H3) and one variable region (V_H). Attached to each heavy chain is a light chain (λ or κ). Based on chemical and biological properties, different regions can be distinguished in the IgG molecule: two antigen binding fragments (obtained as F(ab′)₂ by IdeS treatment; herein referred to as Fab) and a crystallizable fragment (Fc). The structure of IgG is schematically presented in Fig. 1.Open in a separate windowFig. 1.Schematic representation of IgG with the heavy γ chains (dark blue), light chains (lighter blue), and N-glycans. In the top right-hand corner of the Fc and Fab areas, the percentages of galactosylation, sialylation, bisection, and fucosylation are depicted. The inset represents the stable heptasaccharide core with possible extensions.IgGs are glycoproteins, and N-glycans are present at Asn297 of the C_H2 domain. These glycans consist of a constant heptasaccharide core that is often modified by a core fucose and is in part decorated with bisecting N-acetylglucosamine (GlcNAc), galactose(s), and sialic acid(s) (Fig. 1) (1). The Fc glycans have been extensively studied, and glycosylation changes have been found to be associated with disease (e.g. rheumatoid arthritis) (2, 3) and aging (4–6). Several immune regulatory properties have been demonstrated for IgG Fc glycans (7–13). For example, Fc-linked glycans influence the IgG effector function by altering the three-dimensional structure of the protein, and thereby the binding to Fcγ-receptors (12, 13). Additionally, glycan–glycan interactions occur between IgG and Fcγ-receptor-IIIa (8), with the presence of a core fucose decreasing this affinity by ∼2 orders of magnitude (7).The Fab portion consists of the heavy chain C_H1 and V_H regions combined with a light chain and exhibits the antigen binding sites formed by the variable and hypervariable regions of those two chains. N-glycans are known to occur on 15% to 25% of the IgG Fab portions (1, 14, 15). The Fab N-glycans can be involved in immunomodulation, because they influence the affinity and avidity of antibodies for antigens (16–19), as well as antibody half-life (17, 20). The glycans of the Fab have been described as biantennary complex-type structures that are, in contrast to Fc glycans, highly sialylated (21–23). Additionally, high-mannose-type structures have been said to be located on the Fab portion (23).Pregnancy is known to be associated with overall changes in IgG glycosylation. Indeed, a marked increase of galactosylation and sialylation has been observed in IgG Fc glycosylation during pregnancy (3, 24, 25). In addition, lectin binding studies suggest changes in Fab glycosylation of IgG during pregnancy (26), which may be caused by increased levels of progesterone (27). Changes in glycosylation during pregnancy could be one of the mechanisms that contribute to acceptance of the fetal allograft by the maternal immune system (26).Our knowledge on the Fab glycosylation of IgGs from peripheral blood is scarce, which is in part due to difficulty detecting the glycans in a Fab-region-specific manner. Because of the polyclonal nature of serum IgG, one may expect Fab glycans to be attached to a large variety of sequence motifs arising from somatic rearrangements and mutations (28), making the analysis of Fab glycopeptides from polyclonal serum IgG very demanding, if feasible at all. Therefore, study of the Fab glycosylation of polyclonal serum IgG has mainly been pursued at the level of released glycans (14, 23). Difficulties lie in the purification of IgG and the separation of Fc and Fab glycosylation, which is essential for the assignment of the glycans to either part of the IgG molecule.Here we present a high-throughput method for studying Fab glycosylation at the level of released glycans obtained from serum-derived polyclonal IgG. Using state-of-the-art affinity capturing beads and enzymes, we were able to obtain Fab and Fc separately, which, after glycan release, resulted in Fc- and Fab-specific glycan pools. The released glycans were subjected to a novel derivatization protocol resulting in linkage-specific modification of sialic acids, followed by HILIC sample purification and MALDI-TOF-MS. Finally, because marked changes in glycosylation during pregnancy have been described, the technique was applied to consecutive serum samples from a cohort of pregnant women. This approach was chosen to determine the usefulness of this technique in a clinical setting. The method proved to be able to demonstrate pregnancy-related changes in glycosylation of the Fab portion, in addition to the already known changes in Fc glycosylation (3, 24, 25).     

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.     

Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)

Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen''s ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host''s stomach. Although Hp''s glycans are linked to pathogenesis, Hp''s glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp''s glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp''s azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.Helicobacter pylori (Hp)¹ infection poses a significant health risk to humans worldwide. The Gram-negative, pathogenic bacterium Hp colonizes the gastric tract of more than 50% of humans (1). Approximately 15% of infected individuals develop duodenal ulcers and 1% of infected individuals develop gastric cancer (2). Current treatment to clear infection requires “triple therapy” (3), a combination of multiple antibiotics that is often associated with negative side effects (4). Because of poor patient compliance and the evolution of antibiotic resistance, existing antibiotics are no longer effective at eradicating Hp infection (4). New treatment methods are needed to eliminate Hp from the human gastric tract.Recent work has focused on gaining insights into the pathogenesis of Hp to aid the development of new treatments. The most recent findings in this area have conclusively revealed that glycosylation of proteins in Hp is required for pathogenesis. Hp use complex flagella, comprised of flagellin proteins, to navigate the host''s gastric mucosa (5, 6). The flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, found exclusively in mucosal-associated pathogens (Hp (7), Campylobacter jejuni (8) and Pseudomonas aeruginosa (9)). This modification is absolutely essential for the formation of functional flagella on Hp (7, 10). Deletion of any one of the enzymes in the pseudaminic acid biosynthetic pathway results in Hp that lack flagella, are nonmotile, and are unable to colonize the host''s stomach (7). Although pseudaminic acid is critical for Hp virulence, it is absent from humans (11, 12). Therefore, insights into Hp''s pathogenesis have revealed that Hp''s glycan pseudaminic acid is a bona fide target of therapeutic intervention. This is one of a number of examples linking protein glycosylation to virulence in medically significant bacterial pathogens (13, 14).Despite these findings, Hp''s glycome remains poorly understood overall. Only the two flagellin glycoproteins have been firmly characterized in Hp (7) to date. Nine other candidate glycoproteins have been identified in Hp, but their glycosylation status has not been biochemically confirmed (15). The relative paucity of information regarding Hp''s glycoproteins is due in part to the previously held belief that protein glycosylation could not occur in bacteria (13, 16, 17). However, even after Szymanski (18, 19), Koomey (20), Guerry (21), Logan (7), Comstock and others (13, 16, 17) disproved this belief by firmly establishing the synthesis of glycoproteins in bacteria, the study of bacterial glycoproteins has presented unique challenges for analytical study (14, 22). For example, the unusual structures of bacterial glycans, which often contain amino- and deoxy-carbohydrates exclusively found in bacteria (12, 23–25), hampers their identification using existing tools. Though methods such as the use of glycan-binding reagents (20, 24, 26, 27) and periodic acid/hydrazide glycan labeling (15) have successfully detected glycoproteins in a range of bacteria, they present limitations. Glycan binding-based methods are often limited because of the unavailability of lectins or antibodies with binding specificity for glycosylated proteins in the bacteria of interest (14, 22). Periodic acid/hydrazide-based labeling is plagued by a lack of specificity for glycosylated proteins (15). Thus, an efficient and robust approach to discover Hp''s glycoproteins is needed.In previous work, we established that the chemical technique known as metabolic oligosaccharide engineering (MOE), which was developed by Bertozzi (28, 29), Reutter (30), and others for the study of mammalian glycoproteins, is a powerful approach to label and detect Hp''s glycoproteins (31). Briefly, Hp metabolically processes the unnatural, azide-containing sugar peracetylated N-azidoacetylglucosamine (Ac₄GlcNAz) (32), an analog of the common metabolic precursor N-acetylglucosamine (GlcNAc), into cellular glycoproteins (Fig. 1). Elaboration of azide-labeled glycoproteins via Staudinger ligation (33) with a phosphine probe conjugated to a FLAG peptide (Phos-FLAG) (34) followed by visualization with an anti-FLAG antibody (Fig. 1) revealed a glycoprotein fingerprint containing a large number of as-yet unidentified Hp glycoproteins that merit further investigation (31).Open in a separate windowFig. 1.Metabolic oligosaccharide engineering facilitates labeling and detection of Hp''s glycoproteins. Supplementation of Hp with Ac₄GlcNAz leads to metabolic labeling of Hp''s N-linked and O-linked glycoproteins with azides. Azide-modified glycoproteins covalently labeled with Phos-FLAG can be detected via Western blot analysis with anti-FLAG antibody to yield Hp''s glycoprotein fingerprint, which contains a large number of as-yet unidentified glycoproteins.Here we describe a glycoproteomic identification strategy for the selective detection, isolation, and discovery of Hp''s glycoproteins. In particular, we demonstrate that glycan metabolic labeling coupled with mass spectrometry analysis is an efficient and robust chemical approach to identify novel glycoproteins in Hp. This work characterizes glycosylated virulence factors in Hp, thus opening the door to new vaccination and antibiotic therapies to eradicate Hp infection. Broadly, this work validates metabolic oligosaccharide engineering as a complementary method to discover bacterial glycoproteins.     

Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry

Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP1₆₀ (3.36-MDa) and VP1₈₀ (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1₁₈₀). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1₁₈₀ particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP1₆₀ and VP1₈₀ species.Accounting for most cases of non-bacterial gastroenteritis, the norovirus represents an important human pathogen (1, 2). It is the most predominant pathogen within the family Caliciviridae, which also includes Sapovirus, Vesivirus, and Lagovirus (3). The prototypical strain of the genus Norovirus is the Norwalk virus. It is a small (7.7-kb genome) non-enveloped, single-stranded RNA virus. Its genome contains three open reading frames, encoding for the major capsid protein (VP1), the minor capsid protein (VP2), and a non-structural polyprotein (4, 5). VP1 forms homodimers, and the mature Norwalk virus capsids (T = 3) are composed of 90 VP1 dimers (6, 7) and possibly a few copies of VP2 that are thought to stabilize the icosahedral structure as well as affect the expression of VP1 (7, 8). Because of a lack of suitable animal models or in vitro cell culture systems, structural studies so far have been largely focused on recombinant norovirus-like particles (rNVLPs),¹ which are spontaneously assembled during the expression of recombinant VP1 and VP2 in insect cells (5). Importantly, these empty noninfectious particles have been demonstrated to be morphologically and antigenically similar to the genuine virion (9).The rNVLPs have been studied extensively using X-ray crystallography and electron microscopy (EM), which have provided a detailed image of the intact capsid, revealing the T = 3 icosahedral organization (6, 9–11). The VP1 monomer structure is principally composed of two domains, an S domain consisting of the 225 N-terminal residues and a C-terminal P domain. In the intact capsid, the S domain forms a contiguous protein shell with a diameter of ∼30 nm, whereas the P domain forms prominent protrusions, which give the rNVLPs a diameter of ∼40 nm. A remarkable feature of the rNVLPs is that a single protein is responsible for directing capsid assembly and host interactions. The rNVLPs thus represent a simple model to study the assembly of icosahedral viruses. Although the requirements for capsid assembly have been investigated previously (7, 10), there is little information regarding intermediates along the (dis)assembly pathway. Obtaining such information can be quite difficult because of the inherent heterogeneity of capsid assembly. An emerging technique for interrogating such heterogeneous protein assemblies is native electrospray ionization mass spectrometry (ESI-MS).Long regarded as a tool for small molecule analysis and more recently proteomics investigations, the utility of mass spectrometry in structural biology is increasingly applied and accepted (12–15). Native mass spectrometry exploits the gentle ionization conditions afforded by electrospray ionization to transfer intact non-covalently bound protein assemblies into the gas phase. Determining the mass of these complexes with high accuracy allows the oligomeric stoichiometry to be unambiguously deduced. Traditionally challenging targets for structural biology, including complexes in the megadalton range (15–17), heterogeneous or polydisperse assemblies (18, 19), and membrane-bound protein assemblies (20) can now be interrogated in this manner. Furthermore, selective dissociation of these assemblies in both the gas and solution phases allows the designation of subcomplexes, non-covalently bound species smaller than the original protein complex. Combining the knowledge obtained from such data provides information regarding subunit organization at both the architecture and subarchitecture level, allowing the generation of low resolution maps of the overall three-dimensional structure of protein complexes (21–23). Additional information can also be obtained through the combination of tandem MS techniques. CID, for example, can be used to selectively dissociate specific protein assemblies and thus provide information regarding the stability and aid in the assignment of stoichiometry for a given complex. Another tandem MS approach, ion mobility MS (IMMS), provides additional information regarding the shape of gaseous protein complexes. In IMMS, in addition to separation based on their mass-to-charge ratio, ions are also passed through an ion mobility cell with a counterflow of neutral background gas where they are separated based on their size and shape (13, 24).The ability to perform mass measurements of intact viruses has been exploited by several groups but is often limited by mass resolution, which is impeded by the incomplete desolvation of the large protein assemblies during the ionization process. Siuzdak et al. (25) and Robinson and co-workers (26) pioneered the analyses of viruses using mass spectrometry. More recently Uetrecht et al. (15, 17) reported ESI-MS data on the hepatitis B virus (HBV) capsid. In these studies, sufficient mass resolution was obtained to determine the accurate mass and stoichiometry of the T = 3 and T = 4 HBV capsids despite their large mass of 3 and 4 million Da, respectively (15, 17). In addition to being able to measure the mass and stoichiometry of protein assemblies, the capacity of native MS to analyze simultaneously a heterogeneous population of assembly intermediates makes it a powerful technique to study virus assembly (27).In the work described here, the disassembly of rNVLPs was monitored over a range of solution conditions using native ESI-MS, providing insights into their stability and factors that govern icosahedral assembly for this model calicivirus. Unraveling the details of these complex structures and the associated self-assembly pathways that lead to their efficient and precise construction may play an important role in the development of antiviral therapeutics and in the field of nanotechnology where there is much interest in the fundamentals of particle self-assembly.     

A Universal Chemical Enrichment Method for Mapping the Yeast N-glycoproteome by Mass Spectrometry (MS)

Glycosylation is one of the most common and important protein modifications in biological systems. Many glycoproteins naturally occur at low abundances, which makes comprehensive analysis extremely difficult. Additionally, glycans are highly heterogeneous, which further complicates analysis in complex samples. Lectin enrichment has been commonly used, but each lectin is inherently specific to one or several carbohydrates, and thus no single or collection of lectin(s) can bind to all glycans. Here we have employed a boronic acid-based chemical method to universally enrich glycopeptides. The reaction between boronic acids and sugars has been extensively investigated, and it is well known that the interaction between boronic acid and diols is one of the strongest reversible covalent bond interactions in an aqueous environment. This strong covalent interaction provides a great opportunity to catch glycopeptides and glycoproteins by boronic acid, whereas the reversible property allows their release without side effects. More importantly, the boronic acid-diol recognition is universal, which provides great capability and potential for comprehensively mapping glycosylation sites in complex biological samples. By combining boronic acid enrichment with PNGase F treatment in heavy-oxygen water and MS, we have identified 816 N-glycosylation sites in 332 yeast proteins, among which 675 sites were well-localized with greater than 99% confidence. The results demonstrated that the boronic acid-based chemical method can effectively enrich glycopeptides for comprehensive analysis of protein glycosylation. A general trend seen within the large data set was that there were fewer glycosylation sites toward the C termini of proteins. Of the 332 glycoproteins identified in yeast, 194 were membrane proteins. Many proteins get glycosylated in the high-mannose N-glycan biosynthetic and GPI anchor biosynthetic pathways. Compared with lectin enrichment, the current method is more cost-efficient, generic, and effective. This method can be extensively applied to different complex samples for the comprehensive analysis of protein glycosylation.Glycosylation is an extremely important protein modification that frequently regulates protein folding, trafficking, and stability. It is also involved in a wide range of cellular events (1) such as immune response (2, 3), cell proliferation (4), cell-cell interactions (5), and signal transduction (6). Aberrant protein glycosylation is believed to have a direct correlation with the development of several diseases, including diabetes, infectious diseases, and cancer (7–11). Secretory proteins frequently get glycosylated, including those in body fluids such as blood, saliva, and urine (12, 13). Samples containing these proteins can be easily obtained and used for diagnostic and therapeutic purposes. Several glycoproteins have previously been identified as biomarkers, including Her2/Neu in breast cancer (14), prostate-specific antigen (PSA) in prostate cancer (15), and CA125 in ovarian cancer (16, 17), which highlights the clinical importance of identifying glycoproteins as indicators or biomarkers of diseases. Therefore, effective methods for systematic analysis of protein glycosylation are essential to understand the mechanisms of glycobiology, identify drug targets and discover biomarkers.Approximately half of mammalian cell proteins are estimated to be glycosylated at any given time (18). There have been many reports regarding identification of protein glycosylation sites and elucidation of glycan structures (19–30). Glycan structure analysis can lead to potential therapeutic and diagnostic applications (31, 32), but it is also critical to identify which proteins are glycosylated as well as the sites at which the modification occurs. Despite progress in recent years, the large-scale analysis of protein glycosylation sites using MS-based proteomics methods is still a challenge. Without an effective enrichment method, the low abundance of glycoproteins prohibits the identification of the majority of sites using the popular intensity-dependent MS sequence method.About a decade ago, a very beautiful and elegant method based on hydrazide chemistry was developed to enrich glycopeptides. Hydrazide conjugated beads reacted with aldehydes formed from the oxidation of cis-diols in glycans (33). This method has been extensively applied to many different types of biological samples (34–41). Besides the hydrazide-based enrichment method, lectins have also been frequently used to enrich glycopeptides or glycoproteins before MS analysis (28, 29, 42–46). However, there are many different types of lectins, and each is specific to certain glycans (47, 48). Therefore, no combination of lectins can bind to all glycosylated peptides or proteins, which prevents comprehensive analysis of protein glycosylation. Because of the complexity of biological samples, effective enrichment methods are critical for the comprehensive analysis of protein glycosylation before MS analysis.One common feature of all glycoproteins and glycopeptides is that they contain multiple hydroxyl groups in their glycans. From a chemistry point of view, this can be exploited to effectively enrich them. Ideally, chemical enrichment probes must have both strong and specific interactions with multiple hydroxyl groups. The reaction between boronic acids and 1,2- or 1,3-cis-diols in sugars has been extensively studied (49–52) and applied for the small-scale analysis of glycoproteins (53–55). Furthermore, boronate affinity chromatography has been employed for the analysis of nonenzymatically glycated peptides (56, 57). Boronic acid-based chemical enrichment methods are expected to have great potential for global analysis of glycopeptides when combined with modern MS-based proteomics techniques. However, the method has not yet been used for the comprehensive analysis of protein N-glycosylation in complex biological samples (58).Yeast is an excellent model biological system that has been extensively used in a wide range of experiments. Last year, two papers reported the large-scale analysis of protein N-glycosylation in yeast (59, 60). In one study, a new MS-based method was developed based on N-glycopeptide mass envelopes with a pattern via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides with the recoded envelopes were specifically targeted for fragmentation, facilitating high confidence site mapping (59). Using this method, 133 N-glycosylation sites were confidently identified in 58 yeast proteins. When combined with an effective enrichment method, this MS-based analysis will provide a more complete coverage of the N-glycoproteome. The other work combined lectin enrichment with digestion by two enzymes (Glu_c and trypsin) to increase the peptide coverage, and 516 well-localized N-glycosylation sites were identified in 214 yeast proteins by MS (60).Here we have comprehensively identified protein N-glycosylation sites in yeast by combining a boronic acid-based chemical enrichment method with MS-based proteomics techniques. Magnetic beads conjugated with boronic acid were systematically optimized to selectively enrich glycosylated peptides from yeast whole cell lysates. The enriched peptides were subsequently treated with Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F)¹ in heavy-oxygen water. Finally, peptides were analyzed by an on-line LC-MS system. Over 800 protein N-glycosylation sites were identified in the yeast proteome, which clearly demonstrates that the boronic acid-based chemical method is an effective enrichment method for large-scale analysis of protein glycosylation by MS.