Measuring change in glycoprotein structure.

Biosynthetic enzymes in the secretory pathway create distributions of glycans at each glycosite that elaborate the biophysical properties and biological functions of glycoproteins. Because the biosynthetic glycosylation reactions do not go to completion, each protein glycosite is heterogeneous with respect to glycosylation. This heterogeneity means that it is not sufficient to measure protein abundance in omics experiments. Rather, it is necessary to sample the distribution of glycosylation at each glycosite to quantify the changes that occur during biological processes. On the one hand, the use of data-dependent acquisition methods to sample glycopeptides is limited by the instrument duty cycle and the missing value problem. On the other, stepped window data-independent acquisition samples all precursors, but ion abundances are limited by duty cycle. Therefore, the ability to quantify accurately the flux in glycoprotein glycosylation that occurs during biological processes requires the exploitation of emerging mass spectrometry technologies capable of deep, comprehensive sampling and selective high confidence assignment of the complex glycopeptide mixtures. This review summarizes recent technical advances and mass spectral glycoproteomics analysis strategies and how these developments impact our ability to quantify the changes in glycosylation that occur during biological processes. We highlight specific improvements to glycopeptide characterization through activated electron dissociation, ion mobility trends and instrumentation, and efficient algorithmic approaches for glycopeptide assignment. We also discuss the emerging need for unified standards to enable interlaboratory collaborations and effective monitoring of structural changes in glycoproteins.