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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}
}
  • Mass spectrometry-based identification of the components of multiprotein complexes often involves solution-phase proteolytic digestion of the complex. The affinity purification of individual protein complexes often yields nanogram to low-microgram amounts of protein, which poses several challenges for enzymatic digestion and protein identification. We tested different solvent systems to optimize trypsin digestions of samples containing limited amounts of protein for subsequent analysis by LC-MS-MS. Data collected from digestion of 10-, 2-, 1-, and 0.2- g portions of a protein standard mixture indicated that an organicaqueous solvent system containing 80% acetonitrile consistently provided the most complete digestion, producing more peptide identifications thanmore » the other solvent systems tested. For example, a 1-h digestion in 80% acetonitrile yielded over 52% more peptides than the overnight digestion of 1 g of a protein mixture in purely aqueous buffer. This trend was also observed for peptides from digested ribosomal proteins isolated from Rhodopseudomonas palustris. In addition to improved digestion efficiency, the shorter digestion times possible with the organic solvent also improved trypsin specificity, resulting in smaller numbers of semitryptic peptides than an overnight digestion protocol using an aqueous solvent. The technique was also demonstrated for an affinityisolated protein complex, GroEL. To our knowledge, this report is the first using mass spectrometry data to show a linkage between digestion solvent and trypsin specificity. Mass spectrometry (MS) has become a widely used method for studying proteins, protein complexes, and whole proteomes because of innovations in soft ionization techniques, bioinformatics, and chromatographic separation techniques.1-7 An example of a high-throughput mass spectrometry strategy commonly used for this purpose is a variation of the "shotgun" approach, involving in-solution digestion of a protein complex followed by onedimensional (1D) or two-dimensional (2D) liquid chromatography (LC) coupled with electrospray ionization (ESI) MS-MS.6-8 One of the applications of this method is for characterizing multiprotein complexes by identifying large numbers of proteins in a single data acquisition.9 Large-scale implementations of this strategy have been reported for yeast and Escherichia coli.10-12 To achieve a goal of characterizing large numbers of protein complexes13 isolated by affinity purification from Rhodopseudomonas palustris,14 an efficient protocol for digesting these complexes is required.« less
  • A procedure was developed for routine determination of alpha activity resulting from the presence of submicrogram quantities of enriched uranium in urine. Wet oxidation of the organic content, removal of the calcium, and two hours of electrodeposition were found necessary prior to the alpha counting. The method provides over 80% recovery of the alpha activity with a precision of about plus or minus 0.8 counts/minute. (auth)
  • An ion exchange method is given for determination of U both in tungsten- containing minerals and tungsten-free minerals by a column process with high accuracy. Uranium forms a negatively charged chloride complex in a mixture of 20% 4N HCl and 80 to 95% EtOH which is retained on Dowex-1 when most elements are not. Iron(lII) ions present are reduced by ascorbic acid. The U is eluted with ether-saturated 0.1N HCI. (T.R.H.)