A simplified workflow illustrating current glycoproteomic and glycomic analysis strategies. Samples can take the form of slices or spots excised from single or multi-dimensional polyacrylamide gels, batches of cells, fluids, immunoprecipitates or tissue extracts.
Glycoproteomic experiments (blue arrows) can broadly be categorised as “top down” or “bottom up” analyses, with the former beginning with the analysis of the intact glycoproteins, in an attempt to determine the type and extent of glycosylation. Bottom up approaches, essential for describing the glycosylation profiles of proteins in detail, begin with the chemical or enzymatic digestion of the glycoprotein into glycopeptides, followed by mapping experiments carried out either by online nano-LC-ES-MS and MS/MS or offline nano-LC separation followed by subsequent ES or MALDI-TOF/TOF analysis.
Glycomic analyses (green arrows) typically begin with the chemical or enzymatic release of specific pools of glycans. These are then derivatised and subjected to a range of techniques, selected based upon the level of analysis to be carried out – fingerprinting, sequencing, quantification or linkage.
The data produced from these experiments is then interpreted with the assistance of the growing resources of the glycoinformatics community (red arrows) before, ideally, being deposited in raw and annotated form in one of the available databases.

Complementary ‘top down’ and ‘bottom up’ mass spectrometric analysis of the post-translationally modified PilE protein from Neisseria gonorrhoeae. ES-MS ‘top down’ MS analysis of the intact glycosylated protein shows a large cluster of signals corresponding to a series of multiply charged molecular ions. Also observed in the low mass region of the spectrum are sugar oxonium ions, e.g. m/z 229 for the 2,4-diacetamido-2,4,6-trideoxyhexose oxonium ion, which are characteristic of the nature of glycosylation (A). After deconvolution, the ‘top down’ spectra (B) shows the intact glycoprotein to be modified with an array of glycoforms and also differential phosphoethanolaminylation. ES-MS/MS ‘bottom up’ analysis (C) of the PilE glycoprotein after tryptic digestion showing fragmentation of a selected glycopeptide, with site specific glycosylation and phosphoethanolaminylation. The glycan nomenclature is shown in symbol form below.

MALDI-TOF mass spectra of (A) Glycodelin-A (GdA), m/z 1500–4500; (B) Glycodelin-A, m/z 3650 – 3800; (C) bovine pregnancy associated glycoproteins (PAG), m/z 2900 – 5900. Panels A and C cover equivalent mass spans (3000 Da) and for clarity only some of the major signals are annotated. Note that the PAG N-glycan profile is dominated by a single component at m/z 5812.3 [29]. In contrast, the GdA spectrum [28] showed a high level of heterogeneity and more than 130 components are observed over this m/z 3000 range. This is exemplified by the expanded data in Panel B. The peaks which are labelled with “x” are either derived from known contaminants (B) or are derivatisation artefacts (C).

Diagrammatic illustration of the a) O-Fuc and O-Glc glycosylation of the epidermal growth factor (EGF) domains; b) O-Fuc and C-Man glycosylation (linked via carbon, as opposed to oxygen, as indicated in the figure) of TSR-1 domains; c) O-Man sequences are found in mucin domains of proteins such as α-dystroglycan. Sequences can be linear or branched and most are terminated with sialic acid. However sulphated glucuronic acid and fucose can also be present.