Glycan labeling strategies and their use in identification and quantification

Abstract

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed.

Figure

MALDI-FTICR-MS of 2AA-labeled total plasma N-glycans

Fig. 1

Strategies for N-glycan and O-glycan release from glycoproteins and glycopeptides

Fig. 2

Labeling of glycans. a 2-Aminobenzoic acid (2-AA) labeling via reductive amination, b 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition, c labeling with phenylhydrazide, and d glycan permethylation

Fig. 3

Separation of 1-aminopyrene-3,6,8-trisulfonic acid labeled glycans on a microfluidic device in 1 mM phosphate and 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid pH 6.8. Glycans were released from a blood sample of a stage IV breast cancer patient. (Taken from [103] with permission)

Fig. 4

Subfemtomole sensitivity of nanoscale liquid chromatography (LC)–electrospray ionization (ESI)–mass spectrometry (MS) in the analysis of a 2-aminobenzamide (2-AB)-labeled hexamannosidic N-glycan (Man₆GlcNAc₂). A 2-AB-labeled hexamannosidic N-glycan was analyzed using nanoscale hydrophilic interaction LC (HILIC)–ESI–ion trap–MS. Base-peak chromatograms of a dilution series show that the detection of as little as 2 fmol of 2-AB-tagged Man₆GlcNAc₂ injected is possible (A). Sum mass spectra of the elution range of Man₆GlcNAc₂ (25.2–26 min; B–E) revealed the detection of proton adducts (m/z 1,517) and sodium adducts (m/z 1,539) down to 0.5 fmol of injected standard (E). Combined selected ion chromatograms for m/z 1,517 and m/z 1,539 allowed the detection of the standard after injection of a 2-fmol as well as a 0.5-fmol aliquot (inset in A). (Reproduced from [159] with permission)

Fig. 5

Mixed-mode HILIC/anion exchange separation of α₁-acid glycoprotein oligosaccharides before (a) and after (b) desialylation. S1−S5 refer to the number of sialic acid residues present in the oligosaccharides. Bi, Tri, Tri + F, Tetra, and Tetra + F refer to biantennary, triantennary, fucosylated triantennary, tetraantennary, and fucosylated tetraantennary N-linked oligosaccharides, respectively. (Reproduced from [166] from with permission)

Fig. 6

Analysis of N-linked oligosaccharides from human α₁-acid glycoprotein. 2-AB-labeled oligosaccharides obtained were analyzed by high-pH anion-exchange chromatography with fluorescence detection (a) or high-performance LC (HPLC) with an amide-80 column and fluorescence detection (b). (Reproduced from [168] with permission)

Fig. 7

Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans released from a 10 μg RNase B, b 30 μg ovalbumin, and c 30 μg fetuin. The structures of the labeled oligosaccharide peaks are shown in d. Species that were coeluted in the same fluorescence peak are indicated by letters a and b. The peaks corresponding to the species that were eluted early (less than 20 min) containing two or more sialic acids are not shown. Circles mannose, squares GlcNAc, diamonds galactose, triangles fucose, stars sialic acid, dashed lines α linkage, solid lines β linkage, vertical lines 1–2 linkage, horizontal lines 1–4 linkage, forward slashes 1–3 linkage, backslashes 1–6 linkage; wavy lines unknown linkage. (Reproduced from [180] with permission)

Fig. 8

Analysis of 2-AA-labeled total plasma N-glycans by matrix-assisted laser desorption/ionization (MALDI)–Fourier transform ion cyclotron resonance (FTICR)–MS. Samples were prepared as described previously [34], desalted by porous graphitized carbon solid-phase extraction, and analyzed by MALDI-FTICR-MS using a 2,5-dihydroxybenzoic acid matrix. The low-mass range (top) and the high-mass range (bottom) were measured using different ion-transfer times. The inset shows the isotope pattern of registered glycan species. Yellow circles galactose, green circles mannose, blue squares N-acetylglucosamine, purple diamonds sialic acid, red triangles fucose

Fig. 9

A natural glycan microarray approach with reductively aminated glycans. a Glycans are derivatized, b fractionated by HPLC, c analyzed by MALDI time of flight MS(/MS), d immobilized on microarray epoxide slides, e assayed for protein interaction, and f the data obtained are interpreted. (Reproduced from [202] with permission)