The biotinylation of anti-ID antibody was performed using an EZ-LINK biotinylation kit following the manufacturer's protocol. The biotinylated anti-ID antibody was purified using a Zeba 7 kDa molecular weight cut-off desalting spin column, before being stored at -80°C. The final concentration of biotinylated anti-ID antibody was determined to be 8.48 mg/mL. To ensure sufficient capture of ADC1 in plasma incubated samples, the ratio between biotinylated anti-ID antibody and SMART IA magnetic beads was kept at 1:10 (the mass of biotinylated anti-ID antibody in µg to the volume of SMART IA magnetic beads in mL). The tube that contains the solution mixture was rotated for 30 minutes at room temperature using a LabQuake shaker (Barnstead), before loading onto a magnetic tube rack. SMART IA magnetic beads were separated from the solution, and the supernatant was extracted and discarded. The SMART IA beads were washed three times with SN1 buffer (50 mM Tris-HCl in DI water containing 1 mg/mL BSA, pH 7.5) with same volume as the original bead slurry. The anti-ID conjugated SMART IA beads were stored at 4°C until use. 1000 µl of SN1 buffer were added to a new 5 mL Protein LoBind tube, followed by mixing with 300 µL of plasma incubated ADC1 sample. The mixture was briefly vortexed before adding 600 µL conjugated SMART IA beads to the tube. The tube was taped onto a Thermomixer C (Eppendorf) and shaken at 25°C and 1200 rpm for 2 h. After immuno-affinity enrichment, the tube was put onto the magnetic tube rack, and supernatant was discarded. The beads were washed 3 times using 1900 µL of SN2 buffer (50 mM Tris-HCl in DI water, pH 7.5) before 1500 µL SMART IA digestion buffer (provided in the kit) was added to the washed beads. To activate the pre-immobilized trypsin on SMART IA beads, the mixture was shaken at 70°C and 1200 rpm using Thermomixer C (Eppendorf) for 2 h. After tryptic digestion, the tube was placed onto the magnetic tube rack, where digested ADC1 solution was transferred into a new tube. The sample clean-up was performed using an Oasis HLB cartridge (Waters) following the manufacturer's protocol. Post clean-up sample was diluted to 120 µL using 0.1% FA in water, and 30 µL sample was loaded to the LC-MS system.