The lysosomal endopeptidases Cathepsin D and L are selective and effective proteases for the middle‐down characterization of antibodies. (27th March 2021)

• Record Type: Journal Article
• Authors: Čaval, Tomislav; Hecht, Elizabeth Sara; Tang, Wilfred; Uy‐Gomez, Maelia; Nichols, Andrew; Kil, Yong J.; Sandoval, Wendy; Bern, Marshall; Heck, Albert J. R.
• Is Part Of: FEBS journal. Volume 288:Number 18(2021)
• Page Start: 5389
• Page End: 5405
• Publication Date: 2021-03-27
• Subjects: antibodies -- Cathepsin -- high‐resolution native mass spectrometry -- top‐down sequencing -- middle‐down sequencing; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.15813
• Languages: English
• ISSNs: 1742-464X

Abstract

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 sequencing‐sized. Cathepsin D, but not Cathepsin L, also cleaved directly below the Ab hinge, releasing the F(ab')2. When constrained by the different disulfide bonds found in the IgG2 subtype or by the tertiary structure of the hole‐containing bispecific IgG1, the hinge region digest product was not produced. The Cathepsin L and Cathepsin D clipping motifs were related to sequences of neutral amino acids and the tertiary structure of the Ab. A single pot (L + D) digestion protocol was optimized to achieve 100% efficiency. Nine protein fragments, corresponding to the VL, VH, CL, CH1, CH2, CH3, CL + CH1, and F(ab')2, constituted ~ 70% of the summed intensities of all deconvolved proteolytic products. Cleavage sites were confirmed by the Edman degradation and validated with top‐down sequencing. The described work offers a complementary method for middle‐down analysis that may be applied to top‐down Ab sequencing. Enzymes: Cathepsin L—EC 3.4.22.15, Cathepsin D—EC 3.4.23.5 . Abstract : The cleavage sites of the proteases Cathepsins L and D separately, and combined in a one‐pot optimized reaction, were investigated across three antibody types. Cleavages occurred between disulfide bonds and could be prevented by changes in the disulfide pattern (IgG2) or tertiary structure (knob‐and‐hole bispecific). Protein fragments from the digest were ideal‐sized for top‐down sequencing, including the production of a variable region fragments, generated from cleavage directly after the CDR3.