Mapping the proteome of thylakoid membranes by de novo sequencing of intermembrane peptide domains

The proteome of a membrane compartment has been investigated by de novo sequence analysis after tryptic in gel digestion. Protein complexes and corresponding protein subunits were separated by a 2-D Blue Native (BN)/SDS-PAGE system. The transmembrane proteins of thylakoid membranes from a higher plant (Hordeum vulgare L.) were identified by the primary sequence of hydrophilic intermembrane peptide domains using nano ESI-MS/MS-analysis. Peptide analysis revealed that lysine residues of membrane proteins are primarily situated in the intermembrane domains. We concluded that esterification of lysine residues with fluorescent dyes may open the opportunity to label membrane proteins still localized in native protein complexes within the membrane phase. We demonstrate that covalent labelling of membrane proteins with the fluorescent dye Cy3 (TM) allows high sensitive visualization of protein complexes after 2-D BN/SDS-PAGE. We show that pre-electrophoretic labelling of protein subunits supplements detection of proteins by post-electrophoretic staining with silver and CBB and assists in completing the identification of the membrane proteome.