MAR 20, 2019 8:00 AM PDT

Manipulating trypsin digestion conditions to accelerate digestion and improve signal intensity for building a protein-based mass spectrometric assay

Sponsored by: Agilent
C.E. Credits: P.A.C.E. CE Florida CE
Speaker
  • Clinical Chemistry Fellow,Cleveland Clinic
    Biography
      Dr. Yuzi (Emma) Zheng is currently the clinical chemistry fellow at Cleveland Clinic started on July 2017. Dr. Zheng obtained her BSc. in Biochemistry from Concordia University, Montreal, Canada. Then she completed her PhD at the Centre for High-Throughput Biology at University of British Columbia (UBC), Vancouver, Canada; specializing in qualitative and quantitative proteomics. Prior to the clinical chemistry training at Cleveland Clinic, she was a postdoctoral research fellow in the Department of Pathology and Laboratory Medicine at UBC working in the clinical chemistry laboratory at St. Paul's Hospital with research focused on translating biomarker research findings into quantitative mass spectrometry protein assays.

    Abstract

    DATE: March 20, 2019

    TIME:  8:00am PDT

    Multiple aspects need to be considered when building a protein-based mass spectrometric assay such as the selection of signature peptide, calibration matrix and the choice of internal standard (isotopic labeled protein or peptide).  Proteins are reduced to peptides prior to mass spectrometric analysis and protein digestion is a critical step in sample preparation and often a rate limiting step. Trypsin is the most commonly used protease in proteomics experiments; however, digestion can be highly variable and is dependent on several factors including digestion buffer, denaturants, trypsin type, and sample type. Historically, trypsin digestion protocols have relied on lengthy digestion times, which are inappropriate for many clinical research applications. We evaluated numerous iterations of digestion conditions for five plasma proteins and examined which changes yielded the greatest improvement in signal, reproducibility of the digestion profile, and rapid release of proteolytic peptides. It is our hope that this data can help clinical laboratories accelerate the development phase of novel targeted assays by identifying practical approaches to improving digestion protocols.

    Learning objectives:

    • Review the criteria for selecting signature peptide that represent the protein of interest
    • Knowing the different types of peptide digestion profiles
    • Describe the process of peptide selection for building a sensitive and rapid protein-based mass spectrometric assay 

     

     
    For Research Use Only. Not for use in diagnostic procedures.
     
     
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