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Bottoms Up: Improving Proteomic Sample Preparation with Recombinant Asp-N and Multi-Enzyme Workflows

Chris Hosfield is a Senior Research Scientist in the Protein Analysis group at Promega. He has lead the development of tools for proteomics and antibody characterization, including PNGase F, IdeS, IdeZ and recombinant Asp-N.

Chris has broad expertise working with proteolytic enzymes and preparing protein samples for analysis by mass spectrometry. Prior to joining Promega, Chris held senior scientist positions at several biotechnology companies where he applied proteomic methods to the discovery and development of both small molecules and biotherapeutics. He received his PhD in biochemistry from Queen's University.

  • Chris Hosfield, PhD

  • Senior Research Scientist
    Promega Corporation

  • Original Webinar Date: Tuesday, October 16, 2018

While trypsin is the workhorse for bottom-up proteomics, it is not a magic bullet since tryptic digestion often leaves gaps in protein sequences. In this webinar Dr. Hosfield will describe the advantages of using a new recombinant Asp-N protease to fill in the gaps. He will also demonstrate how combining digests from multiple proteases can yield comprehensive sequence coverage of proteins from a single LC-MS/MS run.

Additional Webinar Information:

Bottom-up mass spectrometry workflows typically use trypsin to digest proteins into peptides suitable for LC-MS/MS analysis. While trypsin is an excellent protease, alternative proteases are useful for numerous applications including increasing protein sequence coverage and identifying post-translational modifications.

In this webinar Dr. Hosfield describes the features of a new recombinant Asp-N protease (rAsp-N). rAsp-N displays both high cleavage efficiency and a strong preference for cleavage N-terminal to aspartic acid. He will explain how this enzyme can be effectively used to digest a variety of sample types, including complex protein mixtures and therapeutic molecules such as monoclonal antibodies.

In addition, he describes how a multi-enzyme approach can be successfully employed to achieve 100% sequence coverage of an IgG from a single injection. Combining peptides derived from digestion with orthogonal proteases prior to LC-MS/MS analysis could be a general strategy to ensure high sequence coverage while simultaneously reducing demand on instrument time.