Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and
Hydrophobic Proteins To Enhance Proteome Measurement Depth
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Abstract
The
marine archaeon <i>Nanoarchaeum equitans</i> is dependent
on direct physical contact with its host, the hyperthermophile <i>Ignicoccus hospitalis</i>. As this interaction is thought to
be 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>I. hospitalis-N. equitans</i> proteins. Using this method, 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. 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