Dataset Information

Mass spectrometric analysis of chondroitin sulfate-linked peptides

ABSTRACT: Chondroitin sulfate proteoglycans are integral components of the extracellular matrix. These are composed of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine linked to a serine residue on the core protein through an oligosaccharide linker. The functions of chondroitin sulfate proteoglycans are regulated by the disaccharide heteropolymers attached to the core protein. The degree of sulfation and modifications on the oligosaccharide linker differs in different tissues depending upon its function. Here, we characterized the chondroitin sulfate-linked peptides using mass spectrometric-based glycoproteomics approach. We analyzed plasma, urine and fibroblasts from apparently healthy individuals by mass spectrometry. We identified 230 glycopeptides belonging to 25 chondroitin sulfate-linked proteoglycans.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine, Primary Cell, Blood Plasma, Fibroblast

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD029400 | Pride | 2022-10-13

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	CS_oligosaccharide_linker_compositions.txt	Txt
	FIBRO_Cells_SAX_1600mM_EtHCD.raw	Raw
	FIBRO_Cells_SAX_1600mM_HCD.raw	Raw
	FIBRO_Cells_SAX_800mM_EtHCD.raw	Raw
	FIBRO_Cells_SAX_800mM_HCD.raw	Raw

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Publications

Mass spectrometric analysis of chondroitin sulfate-linked peptides.

Ramarajan Madan Gopal MG Saraswat Mayank M Budhraja Rohit R Garapati Kishore K Raymond Kimiyo K Pandey Akhilesh A

Journal of proteins and proteomics 20221002 4

Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix components composed of linear glycosaminoglycan (GAG) side chains attached to a core protein. CSPGs play a vital role in neurodevelopment, signal transduction, cellular proliferation and differentiation and tumor metastasis through interaction with growth factors and signaling proteins. These pleiotropic functions of proteoglycans are regulated spatiotemporally by the GAG chains attached to the core protein. There are over 70 cho ...[more]

PMID: 36213313

Similar Datasets

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.

Project description:The inter-alpha-trypsin inhibitor (IαI) complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The IαI complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcA3Gal3Gal4Xyl-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the non-sulfated non-reducing end, (GalNAc4GlcA3)n ,to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation (HCD). The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal two GalNAc residues. The fragmentation behavior of CS glycopeptides under variable HCD conditions displays an energy dependency that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.