Analysis of protein glycosylation is essential in order to correlate certain disease types with oligosaccharide structures on proteins. Here, a method for the MS characterization of site-specific... Show moreAnalysis of protein glycosylation is essential in order to correlate certain disease types with oligosaccharide structures on proteins. Here, a method for the MS characterization of site-specific protein glycosylation is presented. Using asialofetuin and fetuin as model substances, a protocol for glycopeptide dissection was developed based on unspecific proteolysis by Proteinase K. The resulting glycopeptides were then resolved by nanoscale hydrophilic interaction liquid chromatography-electrospray multistage MS. The early elution range of O-glycopeptides was clearly separated from the late elution range of N-glycopeptides. Glycopeptides were analyzed by ion trap-MS/MS, which revealed fragmentations of glycosidic linkages and some peptide backbone cleavages; MS3 spectra predominantly exhibited cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N- and O-glycan attachment sites of fetuin could be confirmed; moreover using our method, the occupation of a new, additional O-glycosylation site serine 296 was found. In conclusion, this approach appears to be a valuable technique for in-depth analysis of the site-specific N-glycosylation and O-glycosylation of individual glycoproteins. Show less
Schistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multicellular parasites that live in the mammalian host for many years. Glycoconjugates derived from the... Show moreSchistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multicellular parasites that live in the mammalian host for many years. Glycoconjugates derived from the parasite have been shown to play an important role in many aspects of schistosomiasis, and some of them are present in the circulation of the host. The aim of this study was to identify novel glycoconjugates related to schistosomiasis in urine of Schistosoma mansoni-infected individuals using a combination of glycopeptide separation techniques and in-depth mass spectrometric analysis. Surprisingly, we characterized a heterogeneous population of novel aberrantly O-glycosylated peptides derived from the C terminus of human apolipoprotein C-III (apoC-III) in urine of S. mansoni-infected individuals that were not detected in urine of non-infected controls. The glycan composition of these glycopeptides is completely different from what has been described previously for apoC-III. Most importantly, they lack sialylation and display a high degree of fucosylation. This study exemplifies the potential of mass spectrometry for the identification and characterization of O-glycopeptides without prior knowledge of either the glycan or the peptide sequence. Furthermore, our results indicate for the first time that as a result of S. mansoni infection the glycosylation of a host protein is altered. Molecular & Cellular Proteomics 9: 667-681, 2010. Show less
Scherer, H.U.; Woude, D. van der; Ioan-Facsinay, A.; Bannoudi, H. el; Trouw, L.A.; Wang, J.; ... ; Toes, R.E.M. 2010
OBJECTIVE:: Anti-citrullinated protein antibodies (ACPA) exhibit unique specificity for RA. Whether and how ACPA contribute to disease pathogenesis, however, is incompletely understood. The Fc part... Show moreOBJECTIVE:: Anti-citrullinated protein antibodies (ACPA) exhibit unique specificity for RA. Whether and how ACPA contribute to disease pathogenesis, however, is incompletely understood. The Fc part of human IgG carries two N-linked glycan moieties which are crucial for the structural stability of the antibody and modulate its binding affinity to Fcgamma receptors and its ability to activate complement. We have purified ACPA from serum and synovial fluid and analyzed Fc glycosylation profiles in a specific manner. METHODS:: ACPA were isolated by affinity purification using cyclic citrullinated peptides as antigen. IgG(1) Fc glycosylation was analyzed by mass spectrometry. ACPA glycan profiles were compared to glycan profiles of total serum IgG(1) obtained from 85 well-characterised patients. Glycan profiles of paired synovial fluid and serum samples were available from 11 additional patients. RESULTS:: Compared to the pool of serum IgG(1), ACPA IgG(1) lack terminal sialic acid residues. In synovial fluid, ACPA are highly agalactosylated and lack sialic acid residues, a feature that was not detected for total synovial fluid IgG(1). Moreover, differential ACPA glycan profiles were detected in RF-positive versus -negative patients. CONCLUSION:: ACPA IgG(1) exhibit a specific Fc-linked glycan profile which is distinct from total serum IgG(1). Moreover, Fc glycosylation of ACPA differs markedly between synovial fluid and serum. As Fc glycosylation directly affects the recruitment of Fc-mediated effector mechanisms, these data could further our understanding of the contribution of ACPA to disease pathogenesis. Show less
Selman, M.H.J.; McDonnell, L.A.; Palmblad, M.; Ruhaak, L.R.; Deelder, A.M.; Wuhrer, M. 2010
Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation is essential for Fc-receptor-mediated activities. Changes in IgG Fc glycosylation have been found to be associated with various... Show moreImmunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation is essential for Fc-receptor-mediated activities. Changes in IgG Fc glycosylation have been found to be associated with various diseases. Here we describe a high-throughput IgG glycosylation profiling method. Sample preparation is performed in 96-well plate format: IgGs are purified from 2 mu L of human plasma using immobilized protein A. IgGs are cleaved with trypsin, and the resulting glycopeptides are purified by reversed-phase or hydrophilic interaction solid-phase extraction. Glycopeptides are analyzed by intermediate pressure matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Notably, both dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) matrixes allowed the registration of sialylated as well as nonsialylated glycopeptides. Data were automatically processed, and IgG isotype-specific Fc glycosylation profiles were obtained. The entire method showed an interday variation below 10% for the six major glycoforms of both IgG1 and IgG2. The method was found suitable for isotype-specific high-throughput IgG glycosylation profiling from human plasma. As an example we successfully applied the method to profile the IgG glycosylation of 62 human samples. Show less
Huhn, C.; Ramautar, R.; Wuhrer, M.; Somsen, G.W. 2010
Over the last two decades, coupled capillary electrophoresis (CE)-mass spectrometry (MS) has developed into a generally accepted technique with a wide applicability. A growing number of CE-MS... Show moreOver the last two decades, coupled capillary electrophoresis (CE)-mass spectrometry (MS) has developed into a generally accepted technique with a wide applicability. A growing number of CE-MS applications make use of capillaries where the internal wall is modified with surface coating agents. In CE-MS, capillary coatings are used to prevent analyte adsorption and to provide appropriate conditions for CE-MS interfacing. This paper gives an overview of the various capillary coating strategies used in CE-MS. The main attention is devoted to the way coatings can contribute to a proper CE-MS operation. The foremost capillary coating methods are discussed with emphasis on their compatibility with MS detection. The role of capillary coatings in the control of the electroosmotic flow and the consequences for CE-MS coupling are treated. Subsequently, an overview of reported applications of CE-MS employing different coating principles is presented. Selected examples are given to illustrate the usefulness of the coatings and the overall applicability of the CE-MS systems. It is concluded that capillary coatings can enhance the performance and stability of CE-MS systems, yielding a highly valuable and reproducible analytical tool. Show less
