Mass Spectrometric Analysis of N-Glycoforms of Soybean Allergenic Glycoproteins Separated by SDS-PAGE

Abstract

Glycosylation of many proteins has been revealed to be closely related with food allergies, and screening and structural analysis of related glycoproteins and glycoallergens are essential for studies in this field. Herein, we describe detailed N-glycoform analysis of all glycoprotein fractions of soybean protein isolate (SPI) separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to disclose structural features of the glycan moieties of more soybean glycoproteins. SPI was fractionated by SDS-PAGE, and the generated protein bands were recovered and subjected to in-gel N-glycan release and labeling using a one-pot method newly developed by our group, followed by detailed analysis by electrospray ionization mass spectrometry (ESI-MS) and online hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS). As a result, we found seven bands mainly containing oligomannose-type glycans; two mainly contain core α1,3-fucosylated glycans, and six have no glycans. This study is the first report that discovers core α1,3-fucosylated N-glycans in bands 1, 2, and 6 and discloses bands 3, 4, 5, and 7 as glycoproteins and their N-glycoforms. Therefore, it can expand our knowledge about soybean protein glycosylation and provide significant structural reference for research of soybean allergens.

N‐glycosylation of PD‐1 promotes binding of camrelizumab

“…This experiment was performed as previously reported (Li et al, 2016(Li et al, , 2019. Briefly, 250 ll of water and 200 ll of protein denaturation solution were added to 2 mg of PD-1 proteins obtained from 293T cells, prior to heating at 100°C for 10 min.…”

“…The derived PD-1 N-glycans were collected for further analysis. N-Glycan MS was conducted on an LTQ XL linear ion trap mass spectrometer coupled to an electrospray ion source and an HPLC system (Thermo Scientific, San Diego, CA, USA) using the parameters reported for ESI-MS by (Li et al, 2016). A 50% methanol stream was passed through the Rheodyne loop at a flow rate of 20 ll/min.…”

New applications of advanced instrumental techniques for the characterization of food allergenic proteins

“…Using the bottom-up approach, the application of MALDI-TOF/MS for the identification of potential glycosylation sites of Cor a 11 (hazelnut) has been described (Lauer et al 2004). Similarly, the N-glycoforms of soybean allergenic glycoproteins were identified and quantified by ESI-MS, MS/MS and LC-MS/MS (Li et al 2016). Phosphorylation, hydroxylation, acetylation and methylation are other important PTM associated to the allergenicity of proteins, which have been characterized by MS using the bottom-up approach.…”

Recent update on methodologies for extraction and analysis of soybean seed proteins

“…used high‐resolution MS/MS (HR‐MS/MS) for the identification and structural characterization of oligopeptides from soybean and reported that, owing to the high sensitivity, amino acids can be detected at ≤0.1 µg mL −1 . For example, Li et al . isolated and identified N ‐glycoforms of soybean allergenic glycoproteins using SDS‐PAGE and online hydrophilic interaction liquid chromatography (HILIC) coupled with ESI‐MS/MS.…”

“…Li et al 80 used high-resolution MS/MS (HR-MS/MS) for the identification and structural characterization of oligopeptides from soybean and reported that, owing to the high sensitivity, amino acids can be detected at ≤0.1 μg mL −1 . For example, Li et al 81 isolated and identified N-glycoforms of soybean allergenic glycoproteins using SDS-PAGE and online hydrophilic interaction liquid chromatography (HILIC) coupled with ESI-MS/MS. Cucu et al 82 used MALDI-MS/MS as a fast screening tool for the identification of stable soybean-derived tryptic markers even if the proteins were subjected to various changes on the molecular level through several reactions typically occurring during food processing.…”