Enzymatic Release and MALDI-MS Analysis of N-Glycans

To release the N-glycans from the immobilized proteins, the well module was removed and a M5 TM-Sprayer (HTX Technologies) was used to spray a 0.1 mg/mL PNGase F PRIME solution in water with 15 passes at 25 μL/min, 1200 mm/min, 45 °C, 3 mm spacing between passes with 10 psi nitrogen gas. The slide was incubated for 2 h at 37 °C in a preheated humidity chamber. To apply the MALDI matrix α-cyano-4-hydroxycinnamic acid (CHCA, 7 mg/mL in 50% acetonitrile/0.1% trifluoracetic acid), the same automated sprayer was used with 10 passes at 100 μL/min, 1200 mm/min, 79 °C, 2.5 mm spacing between passes with 10 psi nitrogen gas. For Endo F3 treatment, a mix of 10% Endo F3 and 90% PNGase F Prime was sprayed at the same settings as those described for only PNGase F and all other steps remained the same.^{22 (link),25 (link)}