Analysis of glycans and glycoproteins by mass spectrometry

Abstract

Glycosylation is among the important post-translational modifications that occurs in proteins. In particular the heterogeneity of the glycan moiety can affect a range of physio-chemical properties of a glycoprotein. Thus the increasing interest in glycan function has created a need for sensitive detection and analysis.

Mass spectrometric methods are described herein to obtain structural information from glycans. There are two common ways of characterizing glycans by mass spectrometry. The first method involves releasing glycans from protein, purification followed by mass spectrometric analysis. The second approach involves the analysis of glyopeptides after subjecting the glycoprotein to enzymatic digestion.

The first approach is widely used as a method of getting structural information. In this approach, glycans are usually derivatized using reductive amination to facilitate detection. An improved reductive amination procedure is introduced herein with less toxic reagents. In these experiments NaBH(OAc)3 was used as the reducing agent which is an alternative to the toxic, but extensively used, reducing agent, NaBH3CN.

The glycan release method is very useful in getting structural information about glycans. However if one needs to obtain an understanding about how these glycan structures affect the function of glycoproteins on a molecular level, it is important to characterize the glycans in a glycosylation site-specific manner. The feasibility of site-specific analysis is demonstrated here by characterizing glycans of follicle stimulating hormone in two mammalian species.

Finally, a statistical approach (STEP-statistical test of equivalent pathways), which was developed on the ion trap mass spectrometry is extended to a routinely used instrument, the triple quad mass spectrometer. This method is capable of giving structural information for glycans and peptides. The STEP analysis is based on differentiating first generation product ions from the second-generation product ions in a tandem mass spectrum. This information is useful in getting information about ion genealogy.

Together, the three analytical methods developed herein for glycan and glycoprotein analysis serve as useful tools for future researchers who wish to characterize glycosylation on proteins. Additionally, this thesis provides the first direct comparison of site-specific glycosylation profiles of follicle stimulating hormone, isolated from two different species.