Fragmentation-Free Characterization of Glycopeptides via Ion Mobility

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Haptoglobin is a protein that is produced in the liver, can be found in blood serum, and has four possible sites for glycosylation. Increased haptoglobin levels are a potential indicator of maladies such as anemia and various liver diseases. The glycosylation profiles of haptoglobin may also alter its function, but the biological effect of this is still largely unknown. Therefore, understanding the glycosylation patterns of diseased versus healthy haptoglobin samples may lead to more accurate predictive diagnoses.

Current methods of glycopeptide analysis of haptoglobin are insufficient to address the identification of all possible glycoforms. These conventional methods usually require tandem mass spectrometry (MS/MS) for glycan identification, which can lead to limited glycoform identifications due to similar fragmentation patterns and long reverse-phase liquid chromatography (RPLC) methods as well as co-elution problems.

Herein, we discuss how to leverage high-resolution ion mobility (HRIM) as an orthogonal separation technique for the analysis of haptoglobin glycopeptides ranging from 5-11 AA in length. RPLC-HRIM-MS data and MS/MS fragmentation data were analyzed via Protein Metrics Byos Software (PMI). This study demonstrates that RPLC-HRIM-MS analysis of haptoglobin glycopeptides allows for improved identifications of the glycosylation patterns of glycopeptides than MS/MS fragmentation spectra alone.