Detection of single hinge cleaved trastuzumab and trastuzumab proteolytic cleavage using mass spectrometry. (A) Sequence of the hinge region of trastuzumab showing the major trypsin cleavage sites (black arrows) and the IgG-degrading enzyme S (IdeS) cleavage site (red arrow). Bottom graph: liquid chromatography (LC)/mass spectrometry (MS)/MS analysis of trypsin-treated single hinge cleaved trastuzumab (scIgG-T), identifying the two peptides: THTCPPCPAPELLG and GPSVFLFPPKPK. Spectra are the MS2 spectra showing the b and y ions for the two peptides, confirming their sequences. (B) Sequence of the hinge region of trastuzumab. Black arrows, major trypsin cleavage sites. Bottom graph: LC/MS/MS analysis of trypsin-treated trastuzumab that identifies the peptide THTCPPCPAPELLGGPSVFLFPPKPK. Spectra are the MS2 spectra showing the b and y ions for the peptide, confirming its sequence.