Proteomic Assay Development: Digest Optimization and Quality Control Timothy Collier (Presenter) Quest Diagnostics - Cleveland HeartLab Presenter Bio: Bachelor Degree in Chemistry obtained from NC State University in Raleigh, North Carolina. Continued Graduate School at NC State with a focus in Bioanalytical Chemistry and a Graduate Minor in Biotechnology under the mentorship of Dr. David Muddiman at the W. M. Keck FT-ICR Mass Spectrometry Laboratory, working on biological systems ranging from fungal crop pathogens to human embryonic stem cell biology, with methodological focuses on quantitative Top-Down and Bottom-Up Proteomic Methodologies. After completion of Ph.D. 2010, continued as a Post-Doctoral Research Scholar in the division of Molecular Oncology at Washington University School of Medicine in St. Louis, MO, in the lab of Dr. Ron Bose, and with additional mentorship from Dr. Michael Gross, using quantitative and structural proteomics in breast cancer studies focusing on drug resistance and mutation-driven structural effects

Authors: Timothy S. Collier
Quest Diagnostics - Cleveland HeartLab

The development of protein mass spectrometric assays for use in the clinical laboratory presents unique workflow challenges. Specific pre-analytical steps, such as enzymatic proteolytic digestion, are often required to generate peptides amenable to mass spectrometric analysis. This process requires close attention during and after assay development to ensure reproducible and accurate clinical analysis.

This practical training session will focus on Digest Optimization, including enzyme selection, reagent systems to boost efficiency, and quality control mechanisms to allow monitoring of digest performance. Additional quality control mechanisms to monitor assay performance will also be discussed, including secondary peptide and ion-ratio monitoring, enrichment process monitoring, and steps to ensure reagent quality.

Introduction

The deployment of proteomic assays in the clinical laboratory is in its infancy compared to more traditional immunological-based platforms. Given the wide range of physical and chemical properties of proteins, methods for their analysis can present unique challenges in their development and routine use. Many assays utilize enzymatic digestion workflows to reduce proteins to surrogate peptides that are more amenably measured in a typical LC-MS workflow. . This process requires close attention during and after assay development to ensure reproducible and accurate clinical analysis.

The first part of this practical training session will present a typical digestion workflow and offer insights into pre-digestion sample treatment strategies, enzyme selection, and optimization of digestion conditions.

The second and third parts of this practical training session will discuss strategies for assessing reagent quality and assay performance, both with respect to the development process and to monitoring assay performance in an operations environment.

Methods

Results

Conclusions & Discussion