The diversification of the chemical properties and biological functions of proteins is attained through posttranslational modifications, such as glycosylation. Glycans, which are covalently attached to proteins, play a vital role in cell activities. The microheterogeneity and complexity of glycan structures associated with proteins make comprehensive glycomic analysis challenging. However, recent advancements in mass spectrometry (MS), separation techniques, and sample preparation methods have primarily facilitated structural elucidation and quantitation of glycans. This review focuses on describing recent advances in MS-based techniques used for glycomic analysis (2012-2018), including ionization, tandem MS, and separation techniques coupled with MS. Progress in glycomics workflow involving glycan release, purification, derivatization, and separation will also be highlighted here. Additionally, the recent development of quantitative glycomics through comparative and multiplex approaches will also be described.

Post-translational modifications are vital aspects of functional proteins. Therefore, it is critical to understand their roles in biological processes. Glycosylation is particularly challenging to study among these modifications due to the heterogeneity displayed by the glycans in terms of their isomers. Thus, researchers continue to strive for the development of efficient liquid chromatography techniques for isomeric separation of glycans. Porous graphitized carbon (PGC) nano column has been one of the most widely used columns for this purpose, but poor stability and lack of reproducibility led to its discontinuation. In our endeavor to find an alternative stationary phase for isomeric glycan separation, we tested the mesoporous graphitized carbon (MGC) material. Unprecedentedly, satisfactory results were obtained with a column only 1 cm long, which was tested on permethylated N-glycans derived from model glycoproteins as well as biological samples. The column was found to be reproducible across months as well as across different column preparations. Additionally, to decrease the dead volume and attain a better resolution, MGC was utilized to pack a 1 cm length of a pulled capillary nanospray emitter and again demonstrated efficient isomeric separation. Thus, MGC proved to be a suitable stationary phase to obtain efficient isomeric separation of permethylated N-glycans with 1 cm-long packing length, in both capillary columns and packed nanospray emitters.

Retention time is the most common and widely used criterion to report the separation of glycans using Liquid Chromatography (LC), but it varies widely across different columns, instruments and laboratories....