For pepNde mapping based MAM, GLP-Fc fusion protein DS was diluted to 1 mg/mL with 7.0 M guanidine hydrochloride, 100 mM Tris pH 8.3, to yield a final volume of 100 μL. Two microliters of 500 mM DTT were added to reduce disulphide bonds and allowed to react at room temperature for 30 min under agitaNon. Following reducNon, the samples were alkylated for 20 min at room temperature in the dark by adding 4 μL of 500 mM IAC. AlkylaNon was stopped by adding 4 μL of 50 mM DTT. Buffer exchange was performed using Bio-Spin-6 columns as per the manufacturer's instrucNons. A total of 110 μL of the reduced and alkylated sample was added to the column bed, and the flow-through was collected in a fresh 1.5 mL microcentrifuge tube by centrifugaNon at 1000 ´ g for 4 min. For digesNon, a total of 100 μg of trypsin was reconsNtuted in 100 μL of LC-MS grade water to generate a final protease concentraNon of 1 mg/mL. Trypsin soluNon was added to the samples at a 1:10 (w/w) raNo followed by incubaNon at 37 °C for 30 min with gentle mixing. DigesNon was halted by adding 10% formic acid at a 1:10 (v/v) raNo and subsequently, samples were transferred to individual vials and placed into the autosampler cooled at 5 °C for pepNde mapping analysis. For the iMAM workflow, both using SEC-MS and SCX-MS, the GLP-Fc fusion sample was diluted to 1 mg/mL with MS grade water for analysis.