Journal: ACS Omega

Article Title: Direct MALDI Glycotyping of Glycoproteins toward Practical Subtyping of Biological Samples

doi: 10.1021/acsomega.2c05429

Figure Lengend Snippet: (a) 0,2 A, 2,4 A, and B type ISD fragment position of N -glycans. MALDI–ISD spectrum of RNase B (20 pmol μL –1 ) with (b) 1,5-diaminonaphthalene (DAN)/DHB/Na (12:10:1), (c) DAN/aniline/DHB/Na (2:10:10:1), and (d) DAN/DHB/Na (2:10:1).

Article Snippet: Ribonuclease B (RNase B) from bovine was purchased from New England BioLabs, Inc. (Beverly, MA, USA).

Techniques:

Journal: Nature Communications

Article Title: Mechanism of cooperative N -glycan processing by the multi-modular endoglycosidase EndoE

doi: 10.1038/s41467-022-28722-w

Figure Lengend Snippet: Processing of HM-type N -glycans on RNAse B. Mass spectrometry of RNAse B treated with a no enzyme (negative control) b EndoE-GH18L c EndoE-GH18L E186Q d EndoE-GH18L + EndoE-GH18L E186Q e EndoE-GH20 f EndoE-GH20 E662Q g EndoE-GH20 + EndoE-GH20 E662Q h EndoBT-3987 (positive control) i EndoE j EndoE-GH18L + EndoE-GH20 k EndoE E186Q l EndoE E186Q + EndoE-GH18L m EndoE E662Q n EndoE E662Q + EndoE-GH20. The peaks corresponding to intact RNaseB are numbered based on the glycoforms found in the single glycosylation site of the protein. The retention time for RNAseB was 2.1 min. For mass deconvolution, the following parameters were used in the BioConfirm software; 1000–2400 m/z and 130–160 kDa. The theoretical and observed mass of each annotated peak are in Supplementary Table .

Article Snippet: For the LC-MS enzymatic activity assays, 20 µL reactions were setup containing 1 µM of RNAse B (NEB), Rituximab ± BgaA galactosidase or human transferrin (Sigma–Aldrich)± MvNA sialidase±BgaA galactosidase and 1 µM of enzyme in PBS pH 7.4.

Techniques: Mass Spectrometry, Negative Control, Positive Control, Software

Journal: bioRxiv

Article Title: Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay

doi: 10.1101/2021.06.04.447131

Figure Lengend Snippet: Experimental conditions for optimal limited deglycosylation of bovine RNase B and fetuin by PNGase F were determined. The optimal A ) NP-40 concentration, B ) temperature, C ) and D ) PNGase F:glycoprotein ratio for RNase B and fetuin, respectively, and, E ) and F ) the optimal time for the deglycosylation of RNase B and fetuin, respectively, were assayed by differential migration of the glycoproteins in 15% SDS-PAGE. Fully deglycosylated (+) and fully glycosylated (−) proteins were included for each optimization reaction, denoted by FD and FG, respectively.

Article Snippet: Proof-of-concept and optimization of the LDA method were performed using two standard glycoproteins, bovine RNase B and fetuin (New England Biolabs) by PNGase F (New England Biolabs) under native conditions.

Techniques: Concentration Assay, Migration, SDS Page