The lysosomal endopeptidases Cathepsin D and L are selective and effective proteases for the middle-down characterization of antibodies.

ABSTRACT: Mass spectrometry is gaining momentum as a method of choice to de novo sequence antibodies (Abs). Adequate sequence coverage of the hypervariable regions remains one of the toughest identification challenges by either bottom-up or top-down workflows. Methods that efficiently generate mid-size Ab fragments would further facilitate top-down MS and decrease data complexity. Here, we explore the proteases Cathepsins L and D for forming protein fragments from three IgG1s, one IgG2, and one bispecific, knob-and-hole IgG1. We demonstrate that high-resolution native MS provides a sensitive method for the detection of clipping sites. Both Cathepsins produced multiple, albeit specific cleavages. The Abs were cleaved immediately after the CDR3 region, yielding ~ 12 kDa fragments, that is, ideal seque

SUBMITTER: Caval T 

PROVIDER: S-EPMC8518856 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature