Protein Deglycosylation of Tick Saliva

For protein deglycosylation, 20 µl of tick saliva was incubated under denaturing conditions with a cocktail of α-Gal-free glycosidases (PNGase F, 36 kDa; α-(2-3,6,8,9)-neuraminidase, 69 kDa; O-glycosidase, 180 kDa; β(1-4)-galactosidase, 350 kDa; β-N-acetylglucosaminidase, 140 kDa) that removes both asparagine-linked (N-linked) and serine/threonine-linked (O-linked) oligosaccharides using the EDEGLY enzymatic protein deglycosylation kit (Merck & Co., Inc.) and following the manufacturer’s recommendations [34 (link)]. After deglycosylation, the tick saliva sample was diluted 1:20 in PBS for filtration first through an Amicon 50 kDa unit (Merck & Co., Inc.) to remove deglycosylases except PNGase F, and then the flow-through with tick salivary proteins (most proteins had less than 50 kDa; Fig. 2A) was filtered through the Amicon 3 kDa (Merck & Co., Inc.) to remove buffer and retain proteins.