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Title: Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth

Journal Article · · Analytical Chemistry
 [1];  [2];  [3];  [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); James Madison Univ., Harrisonburg, VA (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
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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.

Research Organization:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
Grant/Contract Number:
AC05-00OR22725
OSTI ID:
1209198
Journal Information:
Analytical Chemistry, Vol. 87, Issue 15; ISSN 0003-2700
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 9 works
Citation information provided by
Web of Science

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Cited By (2)

Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum journal June 2016
A Multiple Protease Strategy to Optimise the Shotgun Proteomics of Mature Medicinal Cannabis Buds journal November 2019

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