By Christoph Kannicht
Christoph Kannicht and a panel of hugely skilled researchers describe with ease reproducible equipment for detecting and reading the posttranslational differences of protein, quite with reference to protein functionality, proteome learn, and the characterization of pharmaceutical proteins. one of the tools offered are these for reading the task of disulfide bond websites in proteins, protein N-glycosylation and protein O-glycosylation, and oligosaccharides current at particular unmarried glycosylation websites in a protein. extra strong innovations facilitate the research of glycosylphosphatidylinositols, lipid ameliorations, protein phosphorylation and sulfation, protein methylation and acetylation, a-amidation, g-glutamate, isoaspartate, and lysine hydroxylation.
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Notes 1. CAUTION: Cyanoborohydride, trifluoroacetic acid, 1-butylamine, THF, acetonitrile, and OPD are toxic and/or flammable. Avoid contact with skin or inhalation. Wear gloves when preparing all solutions. When acetonitrile, phosphoric acid, THF, TFA, 1-butylamine, and cyanoborohydride are not in use they should be stored in an appropriate place. Dispose these chemicals appropriately. 2. The manufacturer’s part numbers for the equipment and reagents are meant as a guide. Equivalent substitutions may be made.
Glass tubes 13 × 100 mm with Teflon-lined screw caps (Reactor Vessels Oxford GlycoSciences, cat no. I-4022). 6. Vacuum centrifuge. 2. Monosaccharide Analysis 1. Neat trifluoroacetic acid (TFA). 2. 1% (w/v) Aqueous sodium acetate solution. 3. 15. National Scientific, BC16NA-BP) (see Note 4). 4. 4% (w/v) Sodium acetate (trihydrate)-2% (w/v) boric acid (granular) in methanol (see Note 3). 5. Anthranilic acid (AA) solution: Weigh approx 45 mg of anthranilic acid (2-amino benzoic acid) into a polypropylene vial.
7. If sequencing of the peptides should be performed, evaporate the obtained glycopeptide mixture to dryness in a vacuum centrifuge, dissolve it in 500 µL of water, and apply it to reversed-phase (RP) HPLC (5). 8. If deglycosylation of the complete glycoprotein only is performed, apply the tryptic digest directly to enzymatic deglycosylation. 2. Enzymatical Deglycosylation with PNGase F 1. Dissolve 50 mU PNGaseF (50–250 U of commercially available enzyme, see Note 1) in 2 mL of volatile deglycosylation buffer and apply it to dialysis by threefold ultrafiltration (see Note 2) in a Centricon microconcentrator tube by centrifugation at 5000g for 90 min at 4°C.