Methods to study glycoproteins at the blood-brain barrier using mass spectrometry

Glycosylation is the most common posttranslational modification of proteins in mammalian cells and is limited mainly to membrane and secreted proteins. Glycoproteins play several key roles in the physiology and pathophysiology of the blood-brain barrier (BBB) and are attractive as diagnostic markers and therapeutic targets for many neurological diseases. However, large-scale glycoproteomic studies of the BBB have been lacking, largely due to the complexity of analyzing glycoproteins and a lack of available tools for this analysis. Recent development of the hydrazide capture method and significant advances in mass spectrometry (MS)-based proteomics over the last few years have enabled selective enrichment of glycoproteins from complex biological samples and their quantitative comparisons in multiple conditions. In this chapter, we describe methods for: (1) isolating membrane and secreted proteins from BEC and other cells of the neurovascular unit, (2) enriching glycoproteins using hydrazide capture, and (3) performing label-free quantitative proteomics to identify differential glycoprotein expression in various biological conditions. Hydrazide capture, when coupled with label-free quantitative proteomics, is a reproducible and sensitive method that allows for quantitative profiling of a large number of glycoproteins from biological samples for the purposes of differential expression measurements and biomarker discovery.