Masshunter qualitative analysis with bioconfirm version b 07.00 software

For 319-44 Fab light and heavy chain mass analysis, 319-44 Fab preparations (see above) were reduced at room temperature with 10 mM TCEP for ~ 2 h. 319-44 Fabs (0.3 mg/ml) were deglycosylated with PNGase F (New England BioLabs, Beverly, MA) at 37°C for an hour, per manufacturer’s recommended protocol. After PNGase F treatment, 319-44 Fabs were washed with 0.1% formic acid and 1 mM DTT thru a 10 kDa molecular weight cutoff Amicon^® centrifugal filter (EMD Millipore, Burlington, MA). Samples were flash frozen after the washing step. 319-44_WT and 319-44_N54A were reduced with 3 M guanidine containing 0.5 M TCEP•HCl and incubated at 37 °C for 3.5 h.
For mass analysis of Fabs, 30 pmol aliquots of sample were trapped and desalted using a C-8 or trap (Zorbax 300SB, 2.1 x 12.5 mm, 5 μm particles) and then separated using a C-8 column (Zorbax 300SB 2.1 × 50 mm, 3.5 μm particle diameter, Agilent, Santa Clara, CA) using a water-acetonitrile gradient. For mass analysis of the mAbs, a C-3 trap and C-4 column having the same characteristics, as the trap and column used for Fab analysis were used. The masses were measured using a quadrupole-time of flight mass spectrometer mass spectrometer (Agilent 6530 in ESI-positive ion mode). Agilent MassHunter Qualitative Analysis with BioConfirm (version B.07.00) software was used for the analysis of all the mass spectrometry data.