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Title: Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples

Abstract

Standard biochemical techniques that are used for protein enrichments, such as affinity isolation and density gradient centrifugation, frequently yield high nanogram to low microgram quantities at a significant expenditure of resources and time. The characterization of selected protein enrichments by the "shotgun" mass spectrometry approach is often compromised by the lack of effective and efficient in-solution proteolysis protocols specifically tailored for these small quantities of proteins. This study compares the results of five different digestion protocols that were applied to 2.5 g portions of protein isolates from two disparate sources: Rhodopseudomonas palustris 70S ribosomal proteins, and Bos taurus microtubule-associated proteins (MAPs). Proteolytic peptides produced according to each protocol in each type of protein isolate were analyzed by one-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). The effectiveness of each digestion protocol was assessed on the basis of three parameters: number of peptide identifications, number of protein identifications, and sequence coverage. The two protocols using a solvent containing 80% acetonitrile (CH3CN) for trypsin digestions performed as well as, and in some instances better than, protocols employing other solvents and chaotropes in both types of protein isolates. A primary advantage of the 80% CH3CN protocol is that it requires fewer sample manipulation steps.

Authors:
 [1];  [1];  [2];  [3]
  1. ORNL
  2. National Institutes of Health
  3. {Greg} B [ORNL
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
930914
DOE Contract Number:
DE-AC05-00OR22725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Proteome Research; Journal Volume: 6; Journal Issue: 8
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ACETONITRILE; AFFINITY; CENTRIFUGATION; DIGESTION; EXPENDITURES; MASS SPECTROSCOPY; PEPTIDES; PROTEINS; PROTEOLYSIS; RHODOPSEUDOMONAS; SOLVENTS; TRYPSIN; trypsin; digestion; proteolysis; acetonitrile; mass spectrometry; liquid chromatography; ribosome; microtubule-associated protein

Citation Formats

Hervey, IV, William Judson, Strader, Michael B, Strader, Michael Brad, and Hurst, Gregory. Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples. United States: N. p., 2007. Web. doi:10.1021/pr070159b.
Hervey, IV, William Judson, Strader, Michael B, Strader, Michael Brad, & Hurst, Gregory. Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples. United States. doi:10.1021/pr070159b.
Hervey, IV, William Judson, Strader, Michael B, Strader, Michael Brad, and Hurst, Gregory. Mon . "Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples". United States. doi:10.1021/pr070159b.
@article{osti_930914,
title = {Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples},
author = {Hervey, IV, William Judson and Strader, Michael B and Strader, Michael Brad and Hurst, Gregory},
abstractNote = {Standard biochemical techniques that are used for protein enrichments, such as affinity isolation and density gradient centrifugation, frequently yield high nanogram to low microgram quantities at a significant expenditure of resources and time. The characterization of selected protein enrichments by the "shotgun" mass spectrometry approach is often compromised by the lack of effective and efficient in-solution proteolysis protocols specifically tailored for these small quantities of proteins. This study compares the results of five different digestion protocols that were applied to 2.5 g portions of protein isolates from two disparate sources: Rhodopseudomonas palustris 70S ribosomal proteins, and Bos taurus microtubule-associated proteins (MAPs). Proteolytic peptides produced according to each protocol in each type of protein isolate were analyzed by one-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). The effectiveness of each digestion protocol was assessed on the basis of three parameters: number of peptide identifications, number of protein identifications, and sequence coverage. The two protocols using a solvent containing 80% acetonitrile (CH3CN) for trypsin digestions performed as well as, and in some instances better than, protocols employing other solvents and chaotropes in both types of protein isolates. A primary advantage of the 80% CH3CN protocol is that it requires fewer sample manipulation steps.},
doi = {10.1021/pr070159b},
journal = {Journal of Proteome Research},
number = 8,
volume = 6,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}