School of BioSciences - Research Publications
Permanent URI for this collection
Search Results
1 - 4 of 4
-
ItemCharacterization of protein N-glycosylation by tandem mass spectrometry using complementary fragmentation techniques(FRONTIERS MEDIA SA, 2015-08-28)The analysis of post-translational modifications (PTMs) by proteomics is regarded as a technically challenging undertaking. While in recent years approaches to examine and quantify protein phosphorylation have greatly improved, the analysis of many protein modifications, such as glycosylation, are still regarded as problematic. Limitations in the standard proteomics workflow, such as use of suboptimal peptide fragmentation methods, can significantly prevent the identification of glycopeptides. The current generation of tandem mass spectrometers has made available a variety of fragmentation options, many of which are becoming standard features on these instruments. We have used three common fragmentation techniques, namely CID, HCD, and ETD, to analyze a glycopeptide and highlight how an integrated fragmentation approach can be used to identify the modified residue and characterize the N-glycan on a peptide.
-
ItemThree UDP-xylose transporters participate in xylan biosynthesis by conveying cytosolic UDP-xylose into the Golgi lumen in Arabidopsis(OXFORD UNIV PRESS, 2018-02-20)UDP-xylose (UDP-Xyl) is synthesized by UDP-glucuronic acid decarboxylases, also termed UDP-Xyl synthases (UXSs). The Arabidopsis genome encodes six UXSs, which fall into two groups based upon their subcellular location: the Golgi lumen and the cytosol. The latter group appears to play an important role in xylan biosynthesis. Cytosolic UDP-Xyl is transported into the Golgi lumen by three UDP-Xyl transporters (UXT1, 2, and 3). However, while single mutants affected in the UDP-Xyl transporter 1 (UXT1) showed a substantial reduction in cell wall xylose content, a double mutant affected in UXT2 and UXT3 had no obvious effect on cell wall xylose deposition. This prompted us to further investigate redundancy among the members of the UXT family. Multiple uxt mutants were generated, including a triple mutant, which exhibited collapsed vessels and reduced cell wall thickness in interfascicular fiber cells. Monosaccharide composition, molecular weight, nuclear magnetic resonance, and immunolabeling studies demonstrated that both xylan biosynthesis (content) and fine structure were significantly affected in the uxt triple mutant, leading to phenotypes resembling those of the irx mutants. Pollination was also impaired in the uxt triple mutant, likely due to reduced filament growth and anther dehiscence caused by alterations in the composition of the cell walls. Moreover, analysis of the nucleotide sugar composition of the uxt mutants indicated that nucleotide sugar interconversion is influenced by the cytosolic UDP-Xyl pool within the cell. Taken together, our results underpin the physiological roles of the UXT family in xylan biosynthesis and provide novel insights into the nucleotide sugar metabolism and trafficking in plants.
-
ItemEnrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings(Humana Press, 2017)The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.
-
ItemN-linked Glycan Micro-heterogeneity in Glycoproteins of Arabidopsis(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2018-03)N-glycosylation is one of the most common protein post-translational modifications in eukaryotes and has a relatively conserved core structure between fungi, animals and plants. In plants, the biosynthesis of N-glycans has been extensively studied with all the major biosynthetic enzymes characterized. However, few studies have applied advanced mass spectrometry to profile intact plant N-glycopeptides. In this study, we use hydrophilic enrichment, high-resolution tandem mass spectrometry with complementary and triggered fragmentation to profile Arabidopsis N-glycopeptides from microsomal membranes of aerial tissues. A total of 492 N-glycosites were identified from 324 Arabidopsis proteins with extensive N-glycan structural heterogeneity revealed through 1110 N-glycopeptides. To demonstrate the precision of the approach, we also profiled N-glycopeptides from the mutant (xylt) of β-1,2-xylosyltransferase, an enzyme in the N-glycan biosynthetic pathway. This analysis represents the most comprehensive and unbiased collection of Arabidopsis N-glycopeptides revealing an unsurpassed level of detail on the micro-heterogeneity present in N-glycoproteins of Arabidopsis. Data are available via ProteomeXchange with identifier PXD006270.