Domain Mapping of Chondroitin/Dermatan Sulfate Glycosaminoglycans Enables Structural Characterization of Proteoglycans

Autor: Jonas Nilsson, Mahnaz Nikpour, Göran Larson, Andrea Persson, Egor Vorontsov
Rok vydání: 2020
Předmět:
Special Issue: Glycoproteomics
IAPP
islet amyloid polypeptide
Iduronic acid
GAG
glycosaminoglycan
Biochemistry
HexA
hexuronic acid
Analytical Chemistry
higher-energy collision dissociation (HCD)
glycomics
Glycosaminoglycan
chemistry.chemical_compound
GalNAc
N-acetylgalactosamine
CgA
chromogranin-A
CS/DS
chondroitin/dermatan sulfate
HA
hyaluronic acid
0303 health sciences
glycan
NRE
nonreducing end
biology
030302 biochemistry & molecular biology
Chondroitin Sulfates
Heparan sulfate
GlcA
glucuronic acid
Proteoglycans
Glycan
HS
heparan sulfate
Dermatan Sulfate
chondroitin/dermatan sulfate (CS/DS)
IdoA
iduronic acid
Xyl
xylose
NCE
normalized collision energy
Dermatan sulfate
Glycomics
03 medical and health sciences
Cell Line
Tumor
TIC
total ion chromatogram
dp
degree of polymerization
DBA
dibutylamine
Chondroitin
Animals
mass spectrometry (MS)
LC-MS/MS
Molecular Biology
030304 developmental biology
Research
PG
proteoglycan
Neu5Ac
N-acetylneuraminic acid
Rats
Gal
galactose
HCD
higher-energy collision dissociation
XIC
extracted ion chromatogram
chemistry
Proteoglycan
biology.protein
Zdroj: Molecular & Cellular Proteomics : MCP
ISSN: 1535-9484
Popis: Of all posttranslational modifications known, glycosaminoglycans (GAGs) remain one of the most challenging to study, and despite the recent years of advancement in MS technologies and bioinformatics, detailed knowledge about the complete structures of GAGs as part of proteoglycans (PGs) is limited. To address this issue, we have developed a protocol to study PG-derived GAGs. Chondroitin/dermatan sulfate conjugates from the rat insulinoma cell line, INS-1832/13, known to produce primarily the PG chromogranin-A, were enriched by anion-exchange chromatography after pronase digestion. Following benzonase and hyaluronidase digestions, included in the sample preparation due to the apparent interference from oligonucleotides and hyaluronic acid in the analysis, the GAGs were orthogonally depolymerized and analyzed using nano-flow reversed-phase LC-MS/MS in negative mode. To facilitate the data interpretation, we applied an automated LC-MS peak detection and intensity measurement via the Proteome Discoverer software. This approach effectively provided a detailed structural description of the nonreducing end, internal, and linkage region domains of the CS/DS of chromogranin-A. The copolymeric CS/DS GAGs constituted primarily consecutive glucuronic-acid-containing disaccharide units, or CS motifs, of which the N-acetylgalactosamine residues were 4-O-sulfated, interspersed by single iduronic-acid-containing disaccharide units. Our data suggest a certain heterogeneity of the GAGs due to the identification of not only CS/DS GAGs but also of GAGs entirely of CS character. The presented protocol allows for the detailed characterization of PG-derived GAGs, which may greatly increase the knowledge about GAG structures in general and eventually lead to better understanding of how GAG structures are related to biological functions.
Graphical Abstract
Highlights • Protocol developed to structurally characterize glycosaminoglycans of proteoglycans. • Comprehensive characterization of cellular glycosaminoglycan structures. • Relative quantification of nonreducing end, internal, and linkage region domains. • Overall chondroitin/dermatan sulfate glycosaminoglycan structures of chromogranin-A.
In Brief Glycosaminoglycans (GAGs) remain one of the most challenging posttranslational modifications to study, much due to their structural complexity and heterogeneity, and new methods for analysis are therefore required. We have developed a protocol for enrichment and structural characterization of GAGs of proteoglycans using nLC-MS/MS. We provide detailed information on the nonreducing end, internal, and linkage region GAG domains and use the data to determine an overall GAG structure of chromogranin-A of rat INS-1832/13 cells.
Databáze: OpenAIRE
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