Rapid and efficient glycoprotein identification through microwave‐assisted enzymatic digestion

Abstract

Identification of protein glycosylation sites is analytically challenging due to the diverse glycan structures associated with a glycoprotein. Mass spectrometry (MS)-based identification and characterization of glycoproteins has been achieved predominantly with the bottom-up approach, which typically involves the enzymatic cleavage of proteins to peptides prior to LC/MS or LC/MS/MS analysis. However, the process can be challenging due to the structural variations and steric hindrance imposed by the attached glycans. Alternatives to conventional heating protocols, that increase the rate of enzymatic cleavage of glycoproteins, may aid in addressing these challenges. An enzymatic digestion of a glycoprotein can be accelerated and made more efficient through microwave-assisted digestion. In this paper, a systematic study was conducted to explore the efficiency of microwave-assisted enzymatic (trypsin) digestion (MAED) of glycoproteins as compared with the conventional method. In addition, the optimum experimental parameters for the digestion such as temperature, reaction time, and microwave radiation power were investigated. It was determined that efficient tryptic digestion of glycoproteins was attained in 15 min, allowing comparable if not better sequence coverage through LC/MS/MS analysis. Optimum tryptic cleavage was achieved at 45°C irrespective of the size and complexity of the glycoprotein. Moreover, MAED allowed the detection and identification of more peptides and subsequently higher sequence coverage for all model glycoprotein. MAED also did not appear to prompt a loss or partial cleavage of the glycan moieties attached to the peptide backbones.

LC–MS/MS Identification of the O-Glycosylation and Hydroxylation of Amino Acid Residues of Collagen α-1 (II) chain from Bovine Cartilage

“…To ensure complete enzymatic digestion, microwave digestion was performed at 45°C and 50W for 30 min. 51 Both GluC and tryptic digestion were quenched through the addition of 0.5-μl aliquots of neat formic acid to the samples.…”

Glycoproteins Enrichment and LC-MS/MS Glycoproteomics in Central Nervous System Applications

“…The examples of oxonium ions are m/z 138 (Hex-CH2O-2H2O), 204 (HexNAc), 274 (NeuAc-H 2 O), 366(Hex + HexNAc) and so on 22, 35 . The software tools for glycopeptide identification are GlypID 31, 32 or GlycoFragWork.…”

Glycoprotein Enrichment Analytical Techniques

“…Specifically, it creates fragmentation pattern of peptides or diagnostic ions of glycan moieties with high intensities at low m/z. The diagnostic ions are also referred as oxonium ions (diagnostic ions), such as m/z 138.0551 (HexNAc-2H2O-CH2O), 204.0875 (HexNAc), 274.0976 (NeuAc-H2O), 366.1395 (Hex+HexNAc), 512.1977 (Hex+HexNAc+dHex) and 657.2353 (NeuAc+Hex+HexNAc) (Mayampurath et al, 2011; Segu, Hammad, & Mechref, 2010). Exists of oxonium ions usually indicate the presence of glycopeptide.…”