Table of Contents
International Journal of Proteomics
Volume 2014 (2014), Article ID 125482, 9 pages
http://dx.doi.org/10.1155/2014/125482
Research Article

A Colorimetric Method for Monitoring Tryptic Digestion Prior to Shotgun Proteomics

1ITSI Biosciences, 633 Napoleon Street, Johnstown, PA 15901, USA
2Windber Research Institute, 620 Seventh Street, Windber, PA 15963, USA

Received 22 October 2013; Accepted 23 December 2013; Published 10 February 2014

Academic Editor: David Sheehan

Copyright © 2014 Richard I. Somiari et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Tryptic digestion is an important preanalytical step in shotgun proteomics because inadequate or excessive digestion can result in a failed or incomplete experiment. Unfortunately, this step is not routinely monitored before mass spectrometry because methods available for protein digestion monitoring either are time/sample consuming or require expensive equipment. To determine if a colorimetric method (ProDM Kit) can be used to identify the extent of tryptic digestion that yields the best proteomics outcome, plasma and serum digested for 8 h and 24 h were screened with ProDM, Bioanalyzer, and LC/MS/MS, and the effect of digestion on the number of proteins identified and sequence coverage was compared. About 6% and 16% less proteins were identified when >50% of proteins were digested in plasma and serum, respectively, compared to when ~46% of proteins were digested. Average sequence coverage for albumin, haptoglobin, and serotransferrin after 2 h, 8 h, and 24 h digestion was 52%, 45%, and 45% for serum and 54%, 47%, and 42% for plasma, respectively. This paper reiterates the importance of optimizing the tryptic digestion step and demonstrates the extent to which ProDM can be used to monitor and standardize protein digestion to achieve better proteomics outcomes.