N- and O- linked Glycosylation, Developing Mass Spectrometric Strategies for the Characterisation of Glyco-epitopes

The overall goal of this thesis was to adopt a liquid chromatography-mass spectrometry (LC-MS) platform for glycomic analysis of glycoproteins. In particular we were interested in characterizing complex glycomic samples such as membrane associated N-linked oligosaccharides and the mucin O- linked oligosaccharides with the aim of identifying particular glycosylation traits or glyco-epitopes that could have biological significance. For the manual interpretation of MSn spectra of oligosaccharides, the acquisition of information-rich MSn spectra is essential. The rearrangement of fragment ions during collision induced dissociation (CID) can complicate the interpretation of the MSn and result in the mis-assignment of an oligosaccharide structure. We have shown that the migration of sulfate groups can occur when sulfated oligosaccharides are fragmented by CID in negative ion mode. The migration is promoted by the presence of a mobile proton and the steric availability of the sulfate groups. Fragmentation by high energy C-trap dissociation (HCD) limits the migration of sulfated residues to where it cannot be detected. Our analysis of membrane associated N-linked oligosaccharides showed that the use of semi-quantitative data can be useful tool at highlighting difference in glycosylation and the use of statistical tools such as monosaccharide composition analysis (MSAC) was useful at providing a global overview of the glycosylation or sorting the oligosaccharide according to particular glycosylation traits such as common core extensions or terminal epitopes provided information on the biological aspects of the oligosaccharides. Our characterisation of O-linked oligosaccharides derived from MUC5AC of healthy and tumour-associated tissue revealed the presence of the novel lacdiNAc (GalNAc Beta 1-4GlcNAc) epitope on gastric mucin. We identified the lacdiNAc epitope by comparing the MS3 of the lacdiNAc containing structure to known standards with terminal N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) residues and by digestion of the lacdiNAc containing oligosaccharides with exo-N-hexosaminidase. We believe this epitope plays a role in inhibiting the binding and proliferation of Helicobacter pylori to MUC5AC by limiting the synthesis of oligosaccharides displaying Leb and sLex epitopes which are required for the adhesion of H. pylori via its adhesins BabA and SabA respectively

Structural Identification of O-Linked Oligosaccharides Using Exoglycosidases and MSn Together with UniCarb-DB Fragment Spectra Comparison

The availability of specific exoglycosidases alongside a spectral library of O-linked oligosaccharide collision induced dissociation (CID) MS fragments, UniCarb-DB, provides a pathway to make the elucidation of O-linked oligosaccharides more efficient. Here, we advise an approach of exoglycosidase-digestion of O-linked oligosaccharide mixtures, for structures that do not provide confirmative spectra. The combination of specific exoglycosidase digestion and MS2 matching of the exoglycosidase products with structures from UniCarb-DB, allowed the assignment of unknown structures. This approach was illustrated by treating sialylated core 2 O-linked oligosaccharides, released from the human synovial glycoprotein (lubricin), with a α2–3 specific sialidase. This methodology demonstrated the exclusive 3 linked nature of the sialylation of core 2 oligosaccharides on lubricin. When specific exoglycosidases were not available, MS3 spectral matching using standards was used. This allowed the unusual 4-linked terminal GlcNAc epitope in a porcine stomach to be identified in the GlcNAc1-4Galb1–3(GlcNAcb1-6)GalNAcol structure, indicating the antibacterial epitope GlcNAca1–4. In total, 13 structures were identified using exoglycosidase and MSn, alongside UniCarb-DB fragment spectra comparison. UniCarb-DB could also be used to identify the specificity of unknown exoglycosidases in human saliva. Endogenous salivary exoglycosidase activity on mucin oligosaccharides could be monitored by comparing the generated tandem MS spectra with those present in UniCarb-DB, showing that oral exoglycosidases were dominated by sialidases with a higher activity towards 3-linked sialic acid rather than 6-linked sialic acid

Towards reproducible MRM based biomarker discovery using dried blood spots

There is an increasing interest in the use of dried blood spot (DBS) sampling and multiple reaction monitoring in proteomics. Although several groups have explored the utility of DBS by focusing on protein detection, the reproducibility of the approach and whether it can be used for biomarker discovery in high throughput studies is yet to be determined. We assessed the reproducibility of multiplexed targeted protein measurements in DBS compared to serum. Eighty-two medium to high abundance proteins were monitored in a number of technical and biological replicates. Importantly, as part of the data analysis, several statistical quality control approaches were evaluated to detect inaccurate transitions. After implementing statistical quality control measures, the median CV on the original scale for all detected peptides in DBS was 13.2% and in Serum 8.8%. We also found a strong correlation (r = 0.72) between relative peptide abundance measured in DBS and serum. The combination of minimally invasive sample collection with a highly specific and sensitive mass spectrometry (MS) technique allows for targeted quantification of multiple proteins in a single MS run. This approach has the potential to fundamentally change clinical proteomics and personalized medicine by facilitating large-scale studies

Presence of terminal n-acetylgalactosamineβ1-4n-acetylglucosamine residues on o-linked oligosaccharides from gastric muc5ac: involvement in helicobacter pylori colonization?

Isolation of MUC5AC mucins from the gastric mucosa from two secretor individuals (one from normal mucosa from a patient with gastric cancer and one from a control) showed different abilities to bind and induce the proliferation of the Helicobacter pylori strain J99. Analysis of the released O-linked oligosaccharides by LC-MS from these individuals showed a very heterogeneous mixture of species from the cancer patient containing both neutral and sialylated structures, whereas the normal sample showed dominating neutral blood group H terminating structures as well as neutral structures containing the di-N-acetyllactosamine (lacdiNAc) unit GalNAc beta 1-4GlcNAc beta 1- on the C-6 branch of the reducing end GalNAc. The linkage configuration of these epitopes were determined using C-4-specific fragmentation for the GalNAc beta 1-4GlcNAc beta 1- glycosidic linkage, comparison of the MS3 fragmentation with standards for linkage configuration and N-acetylhexosamine type as well as exoglycosidase treatment. It was also shown that the lacdiNAc epitope is present in both human and porcine gastric mucins, indicating that this is an epitope preserved between species. We hypothesize that the termination on gastric MUC5AC with lacdiNAc is in competition with complex glycosylation such as the Le(b) and H type 1 as well as complex sialylated structures. These are epitopes known to bind the H. pylori BabA and SabA adhesins

Presence of terminal n-acetylgalactosamineβ1-4n-acetylglucosamine residues on o-linked oligosaccharides from gastric muc5ac: involvement in helicobacter pylori colonization?

Isolation of MUC5AC mucins from the gastric mucosa from two secretor individuals (one from normal mucosa from a patient with gastric cancer and one from a control) showed different abilities to bind and induce the proliferation of the Helicobacter pylori strain J99. Analysis of the released O-linked oligosaccharides by LC-MS from these individuals showed a very heterogeneous mixture of species from the cancer patient containing both neutral and sialylated structures, whereas the normal sample showed dominating neutral blood group H terminating structures as well as neutral structures containing the di-N-acetyllactosamine (lacdiNAc) unit GalNAc beta 1-4GlcNAc beta 1- on the C-6 branch of the reducing end GalNAc. The linkage configuration of these epitopes were determined using C-4-specific fragmentation for the GalNAc beta 1-4GlcNAc beta 1- glycosidic linkage, comparison of the MS3 fragmentation with standards for linkage configuration and N-acetylhexosamine type as well as exoglycosidase treatment. It was also shown that the lacdiNAc epitope is present in both human and porcine gastric mucins, indicating that this is an epitope preserved between species. We hypothesize that the termination on gastric MUC5AC with lacdiNAc is in competition with complex glycosylation such as the Le(b) and H type 1 as well as complex sialylated structures. These are epitopes known to bind the H. pylori BabA and SabA adhesins

The Proteomic Signature of Intestinal Acute Rejection in the Mouse

Intestinal acute rejection (AR) lacks a reliable non-invasive biomarker and AR surveillance is conducted through frequent endoscopic biopsies. Although citrulline and calprotectin have been suggested as AR biomarkers, these have limited clinical value. Using a mouse model of intestinal transplantation (ITx), we performed a proteome-wide analysis and investigated rejection-related proteome changes that may eventually be used as biomarkers. ITx was performed in allogenic (Balb/C to C57Bl) and syngeneic (C57Bl) combinations. Graft samples were obtained three and six days after transplantation (n = 4/time point) and quantitative proteomic analysis with iTRAQ-labeling and mass spectrometry of whole tissue homogenates was performed. Histology showed moderate AR in all allografts post-transplantation at day six. Nine hundred and thirty-eight proteins with at least three unique peptides were identified in the intestinal grafts. Eighty-six proteins varying by >20% between time points and/or groups had an alteration pattern unique to the rejecting allografts: thirty-seven proteins and enzymes (including S100-A8 and IDO-1) were significantly upregulated whereas forty-nine (among other chromogranin, ornithine aminotransferase, and arginase) were downregulated. Numerous proteins showed altered expression during intestinal AR, several of which were previously identified to be involved in acute rejection, although our results also identified previously unreported proteome changes. The metabolites and downstream metabolic pathways of some of these proteins and enzymes may become potential biomarkers for intestinal AR