Technologies for profiling the proteome and even simple post-translational modifications such as phosphorylation are approaching maturity, but the most abundant post-translational modification, glycosylation, still remains practically unexplored at the proteome scale. This is not for lack of interest but because of a dearth of methods for profiling the enormously complex glycoproteome.

Glycans, complex chains of sugars, are not just energy-storage molecules; the important specific biological roles of protein glycosylation are being increasingly brought to light. Eukaryotic cell-surface proteins are often heavily glycosylated, indicating the importance of these modifications in cell signaling, cell-cell interactions and the immune response, for example.

Cell-surface proteins are heavily glycosylated. Credit: Katie Vicari

There are several methodological issues that make tackling the glycoproteome particularly challenging. As a post-translational process, glycosylation is by definition nontemplated. However, unlike simple post-translational modifications such as phosphorylation, the great diversity of glycan structures makes their analysis exponentially more difficult. One single protein can have tens to hundreds of different glycan attachments. Glycosylated forms of proteins are often found in low abundance in the cell, and the modifications themselves in low stoichiometry.

To date, the glycomics field and the proteomics field have often existed in separate spheres. Glycomics researchers profile glycan structures but ignore the proteins from which they came, and proteomics researchers profile proteins while ignoring the appended glycans. However, the importance of integrating the analyses is being realized; proteomics researchers have recently reported high-throughput methods to detect protein glycosylation sites—though not the glycan structures. Glycomics researchers have been able to characterize all glycans found on single proteins—but not in high throughput.

Mass spectrometry, already a proven technology for proteomics, is likely to also be key for glycoproteomics. The very different chemistries of protein chains and glycan chains present a sequencing challenge, but high-resolution mass spectrometry instruments and newer fragmentation methods are likely to facilitate such analyses. Methods for isolating and separating glycoproteins before mass spectrometry analysis, and bioinformatics approaches for analyzing the complex data resulting from such experiments, are also needed.

We hope to see an abundance of new methods for high-throughput glycoproteomics in the near future. Making sense of the data generated from such approaches to sort out the functional roles of glycosylation, however, will take much longer.