Promass software

Prior to LC/MS analysis, conjugates and metabolites were deglycosylated with PNGase F (NEB, cat#P0704L) under non-denaturing conditions at 37°C overnight. ADCs (500 ng) were loaded into a reverse phase column packed with a polymeric material (Michrom-Bruker, cat# CM8/00920/00). LC/MS analysis was performed using Agilent 1100 series HPLC system, comprising binary HPLC pump, degasser, thermostatted auto sampler, column heater and diode-array detector (DAD), coupled to an Orbitrap Velos Pro (Thermo Scientific) mass spectrometer with electrospray ion source. The resulting mass spectra were deconvoluted using ProMass software (Thermo Fisher Scientific).

Purified SaMazF was extensively dialyzed against water and subsequently further desalted and concentrated using C18 spin columns (Thermo Scientific) according to the manufacturer's instructions except that proteins were eluted with 60 μl of 70% acetonitrile in water containing 0.1% formic acid (v/v). Hundred microliters of this SaMazF sample was further diluted using a 50:50 acetonitrile/water mixture containing 0.1% (v/v) formic acid to an approximate final concentration of 5 μM.
The sample was introduced by off-line infusion using a capillary electrospray at 1.5 μl/min into an LTQ XL mass spectrometer (LTQ XL, Thermo Fisher Scientific). Mass spectra with m/z from 400 to 2000 were acquired in centroid mode. Electrospray source conditions such as ‘source fragmentation’ voltage and the tube lens voltage were optimized to help desolvation but without fragmenting the intact protein. Default values were used for most other data acquisition parameters. The resulting spectra were averaged up to 200 scans and were de-convoluted using ProMass software (Thermo Fisher Scientific).