Qualitative and quantitative analysis of proteolytically digested glycoproteins by mass spectrometry

Abstract

Glycoproteins are a very large and biologically relevant class of proteins that comprise more than 50 % of proteins in the human body. The glycosylation present on proteins, specifically N-linked glycosylation has been shown to be important for a variety of processes including protein folding, protein stability, and cell-cell interactions. Many glycoproteins are currently being considered as therapeutic drug targets. Glycosylation on proteins has also been shown to be altered with the onset of diseases, such as cancer, which has opened up the field of glycoproteomics, which aims to detect glycosylation changes for earlier detection of disease states. Mass spectrometry is a versatile technique that is frequently utilized for the analysis of glycoproteins, and it is particularly useful in the detection of glycosylation present on proteins. Most glycoproteins are prepared for mass spectrometric analysis by performing a protease digestion, followed by either a separation by HPLC or some other technique for enrichment of glycopeptides. In this work, the protease digestion procedure was optimized for maximized protein sequence coverage and detection of N-linked glycopeptides and other post-translational modifications. This method was applied to a recombinant glycoprotein that had never before been fully characterized by mass spectrometry and is a potential protein therapeutic as well as known to play a role in different types of cancer. Furthermore, a mass spectrometric relative quantitation method was developed by creating glycosylation profiles from glycopeptides detected at individual glycosylation sites on different glycoproteins. This method allowed for distinguishing between changes in protein concentration from changes in glycosylation. Lastly, glycoprotein structure and stability was probed by circular dichroism spectroscopy before and after glycan removal on glycoproteins containing high mannose type glycans with the enzyme peptide-N-glycosidase F. Protease digestion and mass spectrometry was performed to ensure that the deglycosylation reaction went to completion.