Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth
Abstract
The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. It is thought that this interaction is membrane-associated, involving a myriad of membrane-anchored proteins; proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitansproteins. We show that proteins with increased hydrophobic character, including membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. Moreover, these gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptidesmore »
- Authors:
-
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); James Madison Univ., Harrisonburg, VA (United States)
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
- Publication Date:
- Research Org.:
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1209198
- Grant/Contract Number:
- AC05-00OR22725
- Resource Type:
- Journal Article: Accepted Manuscript
- Journal Name:
- Analytical Chemistry
- Additional Journal Information:
- Journal Volume: 87; Journal Issue: 15; Journal ID: ISSN 0003-2700
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Giannone, Richard J., Wurch, Louie L., Podar, Mircea, and Hettich, Robert L. Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth. United States: N. p., 2015.
Web. doi:10.1021/acs.analchem.5b01187.
Giannone, Richard J., Wurch, Louie L., Podar, Mircea, & Hettich, Robert L. Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth. United States. https://doi.org/10.1021/acs.analchem.5b01187
Giannone, Richard J., Wurch, Louie L., Podar, Mircea, and Hettich, Robert L. 2015.
"Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth". United States. https://doi.org/10.1021/acs.analchem.5b01187. https://www.osti.gov/servlets/purl/1209198.
@article{osti_1209198,
title = {Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth},
author = {Giannone, Richard J. and Wurch, Louie L. and Podar, Mircea and Hettich, Robert L.},
abstractNote = {The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. It is thought that this interaction is membrane-associated, involving a myriad of membrane-anchored proteins; proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitansproteins. We show that proteins with increased hydrophobic character, including membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. Moreover, these gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.},
doi = {10.1021/acs.analchem.5b01187},
url = {https://www.osti.gov/biblio/1209198},
journal = {Analytical Chemistry},
issn = {0003-2700},
number = 15,
volume = 87,
place = {United States},
year = {Thu Jun 25 00:00:00 EDT 2015},
month = {Thu Jun 25 00:00:00 EDT 2015}
}
Web of Science
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