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Increased De Novo Protein Sequencing Coverage with Optimal Protease Cocktail

Part # PS343

Abstract

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

Sample preparation for complete LC-MS/MS sequencing of a pure protein sample differs substantially from the preparation used for protein identification in complex samples. To sequence a protein de novo, the experimental design should aim to identify each amino acid (a.a.) multiple times within different peptides, instead of relying on only a few peptides per protein for complex protein mixtures. Efforts to increase protein sequence coverage typically rely on using multiple proteases to digest the protein. 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. The data presented here can help guide the choice of proteases for maximum coverage during protein sequencing.

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