Antibody-drug conjugates (ADCs) are a novel class of biopharmaceuticals several of which are now being investigated in clinical studies. In ADCs, potent cytotoxic drugs are coupled via a linker to reactive residues in IgG monoclonal antibodies. Linkage to lysine residues in the IgGs, using N-hydroxysuccinimide (NHS) ester-based chemistry, is one of the possible options. To control drug load and specificity, proper knowledge is required about which lysine residues are most accessible and reactive. Here, we combine native mass spectrometry and bottom-up proteomics to monitor the overall drug load and site-specific lysine reactivity, using NHS-based Tandem Mass Tags. High-resolution Orbitrap native mass spectrometry enables us to monitor and quantify, due to the achieved baseline resolution, the sequential incorporation of up to 69 TMT molecules into human IgGs. Complementary, bottom-up proteomics facilitates the identification of some very reactive “hot-spot” conjugation sites. However, we also identify lysine residues that are highly resistant to chemical labeling. Our integrated approach gives insight into the conjugation properties of IgGs at both the intact protein and residue level, providing fundamental information for controlling drug load and specificity in lysine-linked ADCs.This article is protected by copyright. All rights reserved