Accurate discrimination of leucine and isoleucine residues by combining continuous digestion with multiple MS spectra integration in protein sequence.

Protein de novo sequencing based on tandem mass spectrometry is a crucial technology that enables the identification of peptides without searching databases and assembling unknown sequence proteins, especially for monoclonal antibodies (mAbs). However, the discrimination of leucine (Leu) and isoleucine (Ile) residues in the target protein sequence is still challenging. Herein, we developed an accurate method by continuous digestion with MS-based fragmentation and multiple spectra integration (evaluated by combined verification score, CVS) to distinguish Leu and Ile residues. Continuous digestion promotes the diversity of peptides in order to expose more Leu and Ile at the N-terminal. CVS integrates multiple MS spectra to reduce the interference from noise and co-fragmented ions and improve accuracy. This method successfully resolved all 75 Leu/Ile in bovine serum albumin, especially 3 consecutive Leu/Ile. We further applied the method to analyze trastuzumab and 67 out of the 68 Leu/Ile from the light chain and heavy chain were accurately discriminated, demonstrating the great potential in mAbs sequencing.