Analytical glycobiology at high sensitivity: current approaches and directions

Abstract

This review summarizes the analytical advances made during the last several years in the structural and quantitative determinations of glycoproteins in complex biological mixtures. The main analytical techniques used in the fields of glycomics and glycoproteomics involve different modes of mass spectrometry and their combinations with capillary separation methods such as microcolumn liquid chromatography and capillary electrophoresis. The need for high-sensitivity measurements have been emphasized in the oligosaccharide profiling used in the field of biomarker discovery through MALDI mass spectrometry. High-sensitivity profiling of both glycans and glycopeptides from biological fluids and tissue extracts has been aided significantly through lectin preconcentration and the uses of affinity chromatography.

Figure 1

Multimethodological glycoproteomic workflow. A complex biological sample is first depleted of highly abundant proteins, followed by lectin preconcentration of the glycoproteins. Next, the enriched glycoproteins are further fractionated by reversed-phase liquid chromatography (RPLC). Fractions are enzymatically digested, followed by bottom-up LC-MS/MS for protein identification/quantification. (From Reference .)

Figure 2

MALDI-TOF-MS of permethylated N-glycans enzymatically released from α-1-acid glycoprotein (AGP) that was purified from a 5-μL aliquot of human blood serum utilizing a murine monoclonal antibody (mAb). The serum had been previously depleted of seven highly abundant proteins. The mAb was incubated for 1 h with the depleted serum, and the mAb-AGP complex was then extracted from the mixture with anti-mouse IgG coupled to agarose beads. The beads were thoroughly washed to remove the unbound serum proteins, and the purified mAb-AGP was subsequently eluted from the anti-mouse beads with acetic acid, pH 2.6, prior to enzymatic release of N-glycans with PNGase F. We depict here different multiantennary glycans as cartoon structures, using the symbols established by the Consortium for Function Glycomics (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml).

Figure 3

Glycomic and glycoproteomic analysis of so-called “hyperfucosylated” glycoproteins in pancreatic cyst fluids. MALDI-TOF-MS of permethylated N-glycans A) from m/z 1500-3250 and B) from m/z 3250-5000. C) A label-free quantitative comparison of the glycoproteins overexpressed in the hyperfucosylated fluids (G1) to the non-hyperfucosylated fluids (G2), based on whole-fluid proteomics (“No enrichment”) and lectin-enriched glycoproteomics with Aleuria aurantia lectin (AAL) (“AAL-enriched”), revealed the glycoproteins significantly overexpressed and, most importantly, those that were both overexpressed and highly enriched by AAL. (From Reference .)

Figure 4

Combined tandem MS characterization of the ²³⁶VVLHPNYSQVDIGLIK²⁵¹ glycopeptide from human haptoglobin. A) Collision-induced dissociation (CID) preferentially fragments the biantennary, disialylated complex glycan, whereas B) electron-transfer dissociation (ETD) provides complementary information by fragmenting the glycopeptide backbone, which provides the peptide sequence and elucidates the site-specific attachment of the glycan at Asn241. (From Reference .)

Figure 5

O-linked glycans released by a combined enzymatic/chemical method derived from a 1-μg aliquot of bile salt-stimulated lipase (BSSL). (From Reference .)

Figure 6

N-linked glycomic profile of an ovarian cancer patient. The inset highlights the high-mass region where many important trace-level oligosaccharides are located. (From Reference .)

Figure 7

N-linked glycans, isotopically-labeled through permethylation, comparing different developmental stages of Drosophila melanogaster. The m/z values in red are associated with the larval stage, while those in green indicate an embryonic state

Figure 8

Notched-box plots comparing the different ratios of sialic acid linkages of a fucosylated triantennary trisialylated glycan in a) a lung cancer patients (from Reference 146) and b) prostate cancer patients

Figure 8

Notched-box plots comparing the different ratios of sialic acid linkages of a fucosylated triantennary trisialylated glycan in a) a lung cancer patients (from Reference 146) and b) prostate cancer patients

Figure 9

Fetuin-derived glycans separated by a) LC column packed with 3 μm particles and b) UPLC column using 1.7-μm sorbent. (From Reference .)

Figure 10

Optimized workflow for the preparation of glycan-containing samples prior to a capillary electrophoretic analysis. (From Reference .)

Figure 11

Chip-based electropherogram of N-linked glycans from the serum of a patient with late-stage breast cancer. (From Reference .)

Figure 12

Capillary electropherogram depicting the resolution of some isomeric glycans derived from a murine monoclonal antibody. (From Reference .)

Figure 13

Capillary electropherograms demonstrating an on-line exoglycosidase digestion procedure for carbohydrate structural analysis. (From Reference 228.)