Skyline was used to generate all the combinations of modified peptides and to filter false positive identifications. Each identification, which allowed up to 3 modifications per peptide and maximum 4 miscleavages, went through 5 and 10 ppm of precursor or product ion mass filters, obtained by as much as 4 rounds of exhaustive searching, survived 3 barriers of false positive eliminations, and cross-validated by trypsin and chymotrypsin digests. Dual digestions enhanced the sequence coverage and modification visibility. Up to 54 modifications sites were found and the rank of amino acids’ propensity to react in trastuzumab were: Met> Asn> Leu/Ile> Glu> Arg> Cys(C-C)> His > Trp> Tyr> Phe> Thr> Val. Two potential undiscovered metal binding sites and one previously hypothesized binding site were found differentially modified in H2O2 and/or leachable metals stressed conditions. Modifications on trastuzumab binding interphases (Leu H238, His H272, His H437 and AsnH438) to FcɣRIII, complement protein C1q, and FcRn were observed, which could affect their binding activities or even their corresponding physiological functions. Global characterization assay not only offers a broader and deeper views into IgG1 PTMs, but also shows a more complete picture on how oxidative stresses might potentially impact mAb’s functionalities.