412, P412

Simple protocol for preparation and nLC-MS/MS characterization of proteoglycan linkage region glycopeptides of human plasma, urine and cerebrospinal fluid samples

Fredrik Noborn1, Alejandro Gomez Toledo1, Jonas Nilsson1, Göran Larson1,*

1Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden

Introduction: The structural and functional characterization of proteoglycans (i.e. proteins carrying sulfated glycosaminoglycan chains (GAGs) eg. heparan, chondroitin, dermatan or keratan) has for long been focused on their very long and complex glycan chains. This is adequate since most of the biological effects (e.g. extracellular matrix support, growth factor signaling, differentiation, neurogenesis, morphogenesis etc.) are indeed related to differences in the GAGs. However, there is presently a profound lack of knowledge of the structural variability of the GAG core hexasaccharide called the “Linkage Region” and the exact attachment site(s) of these GAGs to various amino acids along the proteoglycan peptide chains.

Objectives: Develop new tools for structural characterization and quantification of proteoglycans in clinical samples.

Methods: Using a combination of anion exchange chromatography, trypsin digestion and glycan hydrolysis using Chondroitinase ABC, core glycopeptides of bikunin (protein AMBP; inter-alfa-trypsin inhibitor light chain), the simplest chondroitin sulfate proteoglycan (CSPG) of human urine, were  enriched and analyzed by reversed phase nLC-MS/MS in positive mode using CID or HCD Orbitrap instrumentation. The protocol was optimized and then employed for analyses of 1-2 mL of urine, cerebrospinal fluid and plasma samples where various CSPGs were identified and their linkage regions characterized.

Results: Several proteins, known as extracellular and cell surface CSPGs were characterized, but also some new CSPGs were identified. The bikunin peptide 206AVLPQEEEGSGGGQLVTEVTK226 of human urine was characterized in detail and shown to have at least 15 different glycoforms, all carrying a linkage region hexasaccharide, with different substitutions of sulfate/s, phosphate and sialic acid, bound to the Ser215 and in half of the isoforms carrying an additional mucin type sialylated O-glycan on a Thr residue.

Conclusion: Our methodology holds promise as clinical analyses of CSPGs of human samples.

Keywords: Biochemistry, Biomarker development, Method