Thierry Le Bihan1, Paul Taylor1, Zac McDonald1, Qixin Liu1, Jianqiao Shen1, Kathleen Gorospe1, Xin Xu1, Chris Hosfield2, Bin Ma1,3
1Rapid Novor, Inc, Kitchener, Ontario, Canada
2Promega Corporation, Madison, WI
3University of Waterloo, Waterloo, Ontario, Canada

Abstract

In this study, we conducted a large-scale statistical analysis of protein sequencing data from samples digested with multiple proteases to understand the impact of using different combinations of proteases to improve the depth of sequence coverage in the application of de novo protein sequencing. MS data for 166 monoclonal antibodies were compiled for use in this study. Each antibody protein sample was digested separately with different proteases and analyzed by LC-MS/MS. The new Pro/Ala (A/P) protease was tested to characterize NIST and MAB04HC antibody standards. De novo peptide sequencing was performed with Novor search algorithm. Protein sequences were assembled using REmAb®. The assembled mAbs demonstrate that a combination of existing proteases with orthogonal activities significantly increases confidence scores in de novo protein sequencing; however, there is a need for new proteases targeting specific amino acid(s) (a.a.) or a.a. sequences to increase antibody sequencing accuracy.

Key Takeaways

  • A combination of proteases can maximize coverage during LC-MS/MS-based sequencing
  • Other orthogonal approaches also contribute to increase in accuracy for de novo protein sequencing
  • REmAb®’s de novo protein sequencing established protocol includes orthogonal and protease cocktails optimized to delivery highly accurate and full coverage protein sequences

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We Have Sequenced 3000+ Antibodies and We Are Eager to Help You.

Through next generation protein sequencing, Rapid Novor enables timely and reliable discovery and development of novel reagents, diagnostics, and therapeutics. Thanks to our Next Generation Protein Sequencing and antibody discovery services, researchers have furthered thousands of projects, patented antibody therapeutics, and ran the first recombinant polyclonal antibody diagnostics

Talk to our scientists. We have sequenced over 3000 antibodies and we are eager to help you.