Selective Detection of Glycopeptides on Ion Trap Mass Spectrometers

Abstract

Generation of carbohydrate-specific marker ions during LC-ESMS of digested glycoproteins has been demonstrated to be a highly selective and sensitive approach for detection of glycopeptides. In principle, any mass spectrometer can produce and selectively detect carbohydrate marker ions provided that the instrument is capable of collisional excitation in the region prior to the first mass analyzer sufficient to form abundant oxonium ions. This approach has yet to be demonstrated on 3D ion trap mass spectrometers, which have become widely used for proteomic applications. Here we report the successful development and optimization of carbohydrate marker ion detection on a LCQ Deca 3D ion trap utilizing this scan function. Human alpha-1 acid glycoprotein and a therapeutic monoclonal antibody were chosen to illustrate this methodology. Marker ion detection during LC-ESMS facilitated collection of glycopeptide-containing fractions. Analysis of the glycopeptides in these fractions by MS identified the specific glycosylation sites and enabled the prediction of the family of glycoforms at each attachment site. Using these optimized conditions, marker ion detection and glycopeptide analysis could be achieved with as little as 10 pmol of a glycoprotein.