Comprehensive Glycopeptide Profiling in Blood Plasma for Clinical Applications
Melissa Baerenfaenger(1) and Hans JCT Wessels(1), Anouk Suppers(1), Merel Post(1), Alain J van Gool(1), Dirk J Lefeber(1) (1)Translational Metabolic Laboratory, Radboudumc, Nijmegen, the Netherlands
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Melissa Baerenfaenger (Presenter) Radboudumc
Presenter Bio: I work as postdoc researcher in Dirk Lefeber’s “Glycosylation Disorder Group”. My work focuses on the development of mass spectrometry-based techniques for the structural characterization of glycoproteins. Furthermore, I promote the Radboud Consortium for Glycoscience to increase awareness and collaboration in this area of research.
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Background:
Almost all proteins in human blood plasma carry glycan structures that impact not only physical properties like solubility but also protein function. Glycoproteins are regarded as high potential biomarkers in blood plasma since aberrant glycosylation is known to occur in various diseases. At present, the glycosylation status of proteins in blood plasma can be determined via released N glycan profiling or targeted analysis of a single glycoprotein. However, these are only incomplete analyses that do not cover the unique combination of glycosylation of several proteins. Glycopeptide profiling offers the potential to generate site-specific glycosylation profiles for hundreds of proteins in a single experiment. To this end, we have developed an innovative glycopeptide profiling approach for blood plasma.
Methods:
Blood plasma was obtained from healthy individuals and selected patients with congenital disorders of glycosylation (CDG). The plasma samples were digested by trypsin and glycopeptides were enriched using Sepharose material. Enriched glycopeptides were then analysed by LC-MS using C18 reversed phase material for HPLC separation coupled to online MS detection using an ESI-qTOF instrument. In-source charge manipulation was performed using organic solvents (nanoBooster, Bruker Daltonics), among others to enhance signal intensity as glycopeptides often show difficulties in ionization under routine ESI conditions. Data dependent CID MS/MS spectra were recorded with optimized settings that favour glycan- or peptide-moiety fragmentation. Glycan moieties were identified in GlycoQuest (Bruker Daltonics) and peptide moieties in MASCOT (MatrixScience). Matlab-based (MathWorks) scripts were developed to integrate all data and to identify differences in glycosylation by multivariate data analysis.
Results:
More than 10.000 unique deconvoluted monoisotopic features were detected at >75% group count between samples. Results obtained by glycopeptide profiling were validated by comparison with released N-glycan profiling and intact glyco-Transferrin LC-MS. Using multivariate analysis, we were able to unambiguously differentiate healthy individuals from patients with known CDG gene defects. The variable importance in projection was used to identify glycopeptide biomarkers for specific gene defects.
Conclusion:
This approach enables comprehensive glycopeptide profiling in blood plasma for clinical applications.
= Discovery stage.
= Translation stage.
= Clinically available.
