Membrane Proteomics in Gram-Positive Bacteria: Two Complementary Approaches to Target the Hydrophobic Species of Proteins
This protocol represents a detailed instruction how to prepare protein samples in order to raise mass spectrometry-based identification and quantification rates with respect to the challenging class of membrane proteins. This will increase comprehensiveness of global proteome studies on the one hand but could also be of interest for researchers targeting specific membrane proteins or membrane protein sequences on the other hand. The protocol is a composite of two parts, one focusing on the identification of protein sequences exterior to a cellular membrane (loops of integral membrane proteins, peripheral membrane proteins), and the other part targeting primarily protein domains spanning the lipid bilayer. The feasibility of the protocol, as it is described here, was originally shown for the gram-positive pathogenic bacterium Staphylococcus aureus but should be applicable to any kind of membrane protein.
Key wordsIntegral membrane proteins Membrane-spanning domains Membrane protein loops Membrane shaving Proteinase K cleavage Chymotryptic digestion
This work was supported by the German Research Foundation Grant SFB/TR34.
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