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