N glycosidase f pngase f

PSA bands were excised with a scalpel, cut into small pieces (around 1 mm³) and frozen at − 20 °C. PSA N-glycans were extracted from the gel pieces following the previously reported methodology^{42 (link)}. Briefly, gel pieces were washed with 20 mM NaHCO₃ and acetonitrile (MeCN), reduced with 50 mM DTT for 30 min at 60 °C and alkylated with 10 mM iodoacetamide (IAA) for 30 min at RT in the dark. Gel pieces were washed with MeCN and dried in the vacuum centrifuge. PSA N-glycans were released by adding 5 µl of N-glycosidase F (PNGase F) (1,000 units/ml, Roche Diagnostics, Mannheim, Germany) diluted with 20 mM NaHCO₃ or 200 uL PNGase F (500,000 units/ml, New England Biolabs, UK) diluted 1/400 in 20 mM NaHCO₃ to completely cover the gel pieces and then incubated at 37 °C overnight. Glycans were extracted from gel pieces with cycles of washing using water and MeCN and then, completely dried.

Lyophilized, iTRAQ-labeled samples (200 µg peptide) were reconstituted with 1 mL lectin binding buffer (20 mM Tris, 0.3 M NaCl, 1 mM CaCl₂, 1 mM MgCl₂, pH 7.4) and incubated with 2 mg AAL agarose beads (Vector Laboratories, Burlingame, CA, USA) via rotation at room temperature for 1 h. After washing with lectin binding buffer three times and 500 µL ddH₂O twice, AAL pull-down peptides were transferred to a new tube and eluted using 10% AA/30% ACN with shaking at room temperature for 10 min. Eluted peptides were re-lyophilized and dissolved in 50 mM sodium phosphate buffer, pH 7.5, supplemented with 90% H₂¹⁸O and 40 U (1 unit/µL) N-glycosidase F (PNGase F; Roche Applied Science, Mannheim, Germany) and incubated at 37 °C with slight shaking for 20 h. Deglycosylated peptides were desalted with 40 µL C18 resin (source 15RPC, GE Healthcare, Björkgatan, Sweden) and lyophilized for further analysis using 2D-SCX/RP-LC-MS/MS.