1.
Nilsson, Jonas, 1970, et al.
(författare)
Targeting the glycoproteome.
2013
Ingår i: Glycoconjugate journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 30:2, s. 119-36
Tidskriftsartikel (refereegranskat) abstract
Despite numerous original publications describing the structural complexity of N- and O-linked glycans on glycoproteins, only very few answer the basic question of which particular glycans are linked to which amino acid residues along the polypeptide chain. Such structural information is of fundamental importance for understanding the biological roles of complex glycosylations as well as deciphering their non-template driven biosynthesis. This review focuses on presenting and commenting on recent strategies, specifically aimed at identifying the glycoproteome of cultured cells and biological samples, using targeted and global enrichment procedures and utilizing the high resolution power, high through-put capacity and complementary fragmentation techniques of tandem mass spectrometry. The goal is to give an update of this emerging field of protein and glyco-sciences and suggest routes to bridge the data gap between the two aspects of glycoprotein characteristics, i.e. glycan structures and their attachment sites.
2.
Halim, Adnan, et al.
(författare)
LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins.
2013
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:2, s. 573-584
Tidskriftsartikel (refereegranskat) abstract
The GalNAc O-glycosylation on Ser/Thr residues of extracellular proteins has not been well characterized from a proteomics perspective. We previously reported a sialic acid capture-and-release protocol to enrich tryptic N- and O-glycopeptides from human cerebrospinal fluid glycoproteins using nanoLC-ESI-MS/MS with collision-induced dissociation (CID) for glycopeptide characterization. Here, we have introduced peptide N-glycosidase F (PNGase F) pre-treatment of CSF samples to remove the N-glycans facilitating the selective characterization of O-glycopeptides and enabling the use of an automated CID-MS2/MS3 search protocol for glycopeptide identification. We used electron capture and transfer dissociation (ECD/ETD) to pinpoint the glycosylation site(s) of the glycopeptides, identified as predominantly core 1 like HexHexNAc-O- glycans attached to 1-4 Ser/Thr residues. We characterized 108 O-glycosylations and found Pro residues preferentially in the n-1, n+1 and/or n+3 positions in relation to the Ser/Thr attachment site (n). The characterization of glycans and glycosylation sites in glycoproteins from human clinical samples provides a basis for future studies addressing the biological and diagnostic importance of specific protein glycosylations in relation to human disease.
3.
Halim, Adnan, et al.
(författare)
Assignment of Saccharide Identities through Analysis of Oxonium Ion Fragmentation Profiles in LC-MS/MS of Glycopeptides.
2014
Ingår i: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 13:12, s. 6024-32
Tidskriftsartikel (refereegranskat) abstract
Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.
4.
Nilsson, Johanna, et al.
(författare)
Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation.
2012
Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1822:4, s. 493-9
Tidskriftsartikel (refereegranskat) abstract
Glycogenin-1 initiates the glycogen synthesis in skeletal muscle by the autocatalytic formation of a short oligosaccharide at tyrosine 195. Glycogenin-1 catalyzes both the glucose-O-tyrosine linkage and the α1,4 glucosidic bonds linking the glucose molecules in the oligosaccharide. We recently described a patient with glycogen depletion in skeletal muscle as a result of a non-functional glycogenin-1. The patient carried a Thr83Met substitution in glycogenin-1. In this study we have investigated the importance of threonine 83 for the catalytic activity of glycogenin-1. Non-glucosylated glycogenin-1 constructs, with various amino acid substitutions in position 83 and 195, were expressed in a cell-free expression system and autoglucosylated in vitro. The autoglucosylation was analyzed by gel-shift on western blot, incorporation of radiolabeled UDP-(14)C-glucose and nano-liquid chromatography with tandem mass spectrometry (LC/MS/MS). We demonstrate that glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe glycogenin-1. Our results explain the glycogen depletion in the patient expressing only Thr83Met glycogenin-1 and why heterozygous carriers without clinical symptoms show a small proportion of unglucosylated glycogenin-1.
5.
Wang, P., et al.
(författare)
Synthesis aided structural determination of amyloid-beta(1-15) glycopeptides, new biomarkers for Alzheimer's disease
2014
Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1359-7345. ; 50:95, s. 15067-15070
Tidskriftsartikel (refereegranskat) abstract
Unique tyrosine glycosylated amyloid-beta(1-15) glycopeptides were synthesized with well-defined stereochemistry at the glycosidic linkages. Aided by these glycopeptides and tandem mass spectrometry analysis, the naturally existing amyloid-beta glycopeptides, isolated from Alzheimer's disease patients, were determined to contain an alpha-linked N-acetyl galactosamine at the modified tyrosine 10 residue. Glycosylation can significantly impact the properties of amyloid-beta as the glycopeptide has much lower affinity for Cu+ ions.
