Dataset Information

Intact Glycopeptide Analysis of Human Urine

ABSTRACT: Identification of intact glycopeptides in pooled healthy human urine samples

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

SUBMITTER: Sharon Pitteri

LAB HEAD: Sharon Pitteri

PROVIDER: PXD038923 | Pride | 2024-02-21

REPOSITORIES: Pride

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Publications

Identification and characterization of intact glycopeptides in human urine.

Garcia-Marques Fernando F Fuller Keely K Bermudez Abel A Shamsher Nikhiya N Zhao Hongjuan H Brooks James D JD Flory Mark R MR Pitteri Sharon J SJ

Scientific reports 20240214 1

Glycoproteins in urine have the potential to provide a rich class of informative molecules for studying human health and disease. Despite this promise, the urine glycoproteome has been largely uncharacterized. Here, we present the analysis of glycoproteins in human urine using LC-MS/MS-based intact glycopeptide analysis, providing both the identification of protein glycosites and characterization of the glycan composition at specific glycosites. Gene enrichment analysis reveals differences in bi ...[more]

PMID: 38355753

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Project description:Chondroitin sulfate proteoglycans (CSPGs), an important class of carbohydrate-modified proteins, are composed of chondroitin sulfate (CS) polysaccharide(s) attached to different core proteins. CS polysaccharides interact with a multitude of protein ligands to control key events in homeostasis and development. Much effort has been devoted to CS structural characterization, as the specificities of the interactions are considered to be determined by the distribution of sulfate groups along the polysaccharide chains. Owing to their structural complexity however, CS chains are typically characterized after they have been released from their core proteins, which precludes site-specific structural information. Such information would likely assist in assigning CSPG-related functions and provide insights to potential differences in CS structures for different core proteins and their glycosites. Here we utilize a novel glycoproteomic approach for the site-specific sequencing of CS modifications of CSPGs from human urine using a combination of biochemical enrichments, enzymatic digestions, and positive mode nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. Trypsin-derived CS-glycopeptides were partially depolymerized with chondroitinase ABC, yielding peptide-attached CS chains, ranging from 6 - 18 monosaccharides in length. Software-assisted- and manual analysis revealed that certain core proteins carried CS with abundant sulfate modifications, while others carried CS with lower levels of sulfation. Inspection of the amino acid sequences surrounding the attachment sites indicated that acidity of the attachment site motifs influenced the levels of CS sulfation and statistical analysis confirmed this relationship. These results demonstrate attachment site-specific CS polysaccharides of CSPGs in human urine and indicate that this novel method may assist in elucidating the biosynthesis and functional roles of CSPGs in cellular physiology.

			Action	DRS
	AB03_Urine_FT.raw	Raw
	AB03_Urine_FT.raw_20210217.xlsx	Xlsx
	AB03_Urine_Fxn1.raw	Raw
	AB03_Urine_Fxn1.raw_20210217.xlsx	Xlsx
	AB03_Urine_Fxn2.raw	Raw