Protein glycosylation plays a significant role in a range of biological processes from cell-cell communication to regulation and activation of key proteins during disease progression. MS is the preferred method for glycan analysis due to it’s ability to discern fine details in complex solutions. Major challenges include the very low abundance of glycosylated proteins in biological matrices and the largely heterogenous nature of glycosylated protein samples.
Modern glycoproteomic workflows involve sample enrichment methods to maximize signal while minimizing complexity. Multiple fragmentation methods are often used to identify glycosylation sites, determine peptide signatures, and characterize glycan structure and arrangement.
Enrichment kits serve to isolate glycan containing peptides and proteins from complex matrices such as blood or cell lysates, typically through the use of immobilized lectins. Solutions and buffers are optimized to allow high enrichment yields and compatibility with LC separations systems.
High performance MS analysis is performed on an advanced instrument such as the Thermo Scientific Orbitrap Fusion Lumos Tribrid MS. Beyond high resolution and analysis speed, this instrument is compatible with multiple dissociation techniques (CID, HCD, ETD, and EThcD), which combine to provide thorough glycan characterization.
Data processing is very challenging in glycan analysis, as not only is peptide sequencing required, but identification of the glycosylation sites and characterization of the glycan arrangement is also needed. The Proteome Discoverer Software from Thermo Scientific uses proprietary software nodes to integrate, for instance, HCD sugar composition data with ETD/EThcD data for peptide backbone determination. This integration brings several data streams together to make sense of complicated results.
Ongoing challenges include the persistence of sample heterogeneity thereby complicating large scale quantitative analyses. Rather complex enrichment procedures continue to limit full automation of sample prep workflows. Regardless, the utility and performance of MS detection and data analysis solutions may very well drive the field of glycosylation into new and interesting territory.