Discovering the phosphoproteome of the hydrophobic cytochrome c oxidase membrane protein complex.
Many cellular processes are regulated by reversible phosphorylation to change the activity state of proteins. One example is cytochrome c oxidase (COX) with its important function for energy metabolism in the mitochondria. The phosphorylation of this enzyme is a prerequisite for the allosteric ATP-inhibition and therefore necessary to adapt energy production to ATP demand of the cell. Its hydrophobic nature hampers the recognition of phosphorylated amino acids in most subunits of this complex, and as a consequence, only a few phosphorylation sites were identified by mass spectrometry. We describe here a method that enables the analysis of integral membrane proteins by chemical cleavage with cyanogen bromide (BrCN), a method that improves the mass spectrometric detection of hydrophobic proteins. The low abundance of phosphopeptides requires efficient enrichment techniques, such as TiO(2)-based methods. However, this strategy failed in our hands when just BrCN-cleaved peptides were used. Only an additional size-reduction with trypsin produced peptides with optimal properties for enrichment and MS-identification. Another bottleneck was the correct assignment of phosphoserine and phosphothreonine because peptide-ion fragmentation by collision induced dissociation (CID) often results in neutral loss of HPO(3) or H(2)PO(4) from the precursor, decreasing fragmentations that define the peptide sequence and the phosphorylation site. The additional usage of electron transfer dissociation (ETD) as an alternative fragmentation method enabled the precise assignment of the phosphorylated amino acids. In a total of six, new phosphorylation sites of four COX-subunits were identified by this strategy.
Department of Functional Proteomics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany. stefan.helling@rub.de
Stefan
S
Helling
Maik
M
Hüttemann
Bernhard
B
Kadenbach
Rabia
R
Ramzan
Sebastian
S
Vogt
Katrin
K
Marcus
D000818:Animals; D002417:Cattle; D056148:Chromatography, Reverse-Phase; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D008565:Membrane Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D003576:Electron Transport Complex IV; D014357:Trypsin