With the purified peptide in hand, 10 equivalents PADI4_3_H4(2) were incubated with PADI4, and intact mass spectrometry (MS) performed. This confirmed that PADI4_3_H4(2) covalently bound to PADI4 at a single site (Figure ). To evaluate whether the peptides were reacting at the active site cysteine, Cys645, we produced an inactive PADI4 variant in which the Cys645 was substituted with alanine, PADI4 C645A, and performed the same experiment (Figure -S4C, S5). No covalent binding between the peptide and PADI4 C645A was observed by intact-MS, confirming that PADI4_3_H4(2) was reacting exclusively with the active site Cys645.