Rapid and Simple Solid‐Phase Esterification of Sialic Acid Residues for Quantitative Glycomics by Mass Spectrometry

Abstract

A rapid and quantitative method for solid-phase methyl esterification of carboxy groups of various sialylated oligosaccharides has been established. The method employed a triazene derivative, 3-methyl-1-p-tolyltriazene, for facile derivatization of oligosaccharides immobilized onto general solid supports such as Affi-Gel Hz and gold colloidal nanoparticles in a multiwell plate. The workflow protocol was optimized for the solid-phase processing of captured sialylated/unsialylated oligosaccharides separated from crude sample mixtures by chemical ligation. From tryptic and/or PNGase F-digest mixtures of glycoproteins, purification by chemoselective immobilization, esterification and recovery were achieved in the same well of the filter plate within three hours when used in conjunction with "glycoblotting technology" (S.-I. Nishimura, K. Niikura, M. Kurogochi, T. Matsushita, M. Fumoto, H. Hinou, R. Kamitani, H. Nakagawa, K. Deguchi, N. Miura, K. Monde, H. Kondo, High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry: Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The recovered materials were directly applicable to subsequent characterization by mass spectrometric techniques such as MALDI-TOF for large-scale glycomics of both neutral and sialylated oligosaccharides. On-bead/on-gold nanoparticle derivatization of glycans containing sialic acids allowed rapid and quantitative glycoform profiling by MALDI-TOF MS with reflector and positive ion mode. In addition to its simplicity and speed, the method eliminates the use of unfavorable halogenated solvents such as chloroform and dichloromethane or volatile solvents such as diethyl ether and hexane, resulting in a practical and green chemical method for automated robotic adaptation.

Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjects

“…Each haptoglobin b-chain was reductively alkylated and digested with trypsin, and N-glycans were enzymatically liberated from each haptoglobin b-chain using PNGase-F, and purified by a combination of methods previously described, with some modifications. [23][24][25] The sialic acid residue(s) of the N-glycans were methylesterified, 26 and labeled with aoWR. 27 For analyses of glycopeptides having N-linked glycan, purified haptoglobin b-chain (4-8 lg) was dried by centrifuge concentrator (Speed-Vac, Savant), and solubilized with 50 mM ammonium bicarbonate (pH 7.8) at a concentration of 0.2-0.4 mg/ml.…”

“…The released N-linked glycans were purified from a crude digestion mixture by glycoblotting technique through reaction of the reducing end of the carbohydrate with an amino-oxime group or hydrazide group affixed on solid phase. 23 To perform quantitative MALDI-TOF analysis with high sensitivity, the captured glycans were further methylesterified at the carboxyl group of sialic acid residue(s), 26 and were derivatized with aoWR. 27 Spectra of the N-linked glycans obtained from the b-chain of prostate cancer, benign prostate disease and normal subjects are shown in Figure 2.…”

Impact of a Three Amino Acid Deletion in the CH2 Domain of Murine IgG1 on Fc-Associated Effector Functions

“…In brief, IgG samples were reductively alkylated under the presence of detergent, then digested successively with trypsin and peptide N-glycosidase F (Roche Diagnostics), as previously described (19). The digested sample was mixed with a novel hydrazide-functionalized glycoblotting polymer (18) and, following washing of the unbound substances (e.g., peptides, detergent, enzymes), sialic acids were methyl-esterified to render sialylated oligosaccharides chemically equivalent to neutral oligosaccharides, as described (20). The IgG oligosaccharides were finally recovered as derivatives of aoWR (N␣-((aminooxy)acetyl)tryptophanylarginine methyl ester), an oligosaccharide labeling reagent that allows highly sensitive detection on mass spectrometry (MS) (21).…”

Sensitive and Structure-Informative <i>N</i>-Glycosylation Analysis by MALDI-MS; Ionization, Fragmentation, and Derivatization

“…61) Miura et al introduced a unique procedure for methyl esteri cation for derivatizing glycans attached to a solid support. 62) As another type of derivatization, the amidation of sialic acid residues using condensing reagent(s) was introduced by Sekiya et al 63) Amidation with methylamine 64) and acetohydrazide 65) was then investigated as more reliable methods for derivatizing sialic acid residues, regardless of their linkage types.…”