Identification of Proteolysis Products in Protein Therapeutics through TMPP N-Terminal Tagging and Electron Transfer Dissociation Product Triggered Collisional Induced Dissociation Fragmentation

Abstract

Thorough characterization of protein therapeutics is often challenging due to the heterogeneity arising from primary sequence variants, post-translational modifications, proteolytic clipping, or incomplete processing of the signal peptide. Modern mass spectrometry (MS) techniques are now routinely used to characterize such heterogeneous protein populations. Here, we present an LC-MS/MS method using (N-succinimidyloxycarbonylmethyl)-tris (2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to label any free N-terminal α-amines to rapidly and selectively identify proteolytic clipping events. Electron transfer dissociation (ETD) fragmentation of these chemically tagged peptides generates two unique TMPP product ions, TMPP+ and TMPP-Ac-NH₂/c0. The presence of these signature ions following ETD is used to trigger subsequent collisional induced dissociation (CID) fragmentation of the precursor ion. This results in a small subset of CID tandem MS spectra that are used in a customized database search. Using a purified fusion monoclonal antibody (mAb) as an example, we demonstrate how TMPP labeling followed by ETD product ion triggered CID fragmentation is used to accurately identify two undesired clipping sites.

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