Abstract
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 TiO2-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 HPO3 or H2PO4 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.
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Acknowledgments
The authors thank Kathy Pfeiffer and Frederic Brosseron for excellent technical assistance and maintenance of the mass spectrometer (both: Department of Functional Proteomics, Ruhr-Universität Bochum, Bochum, Germany). This work was supported by Forum (Forschungsförderung Ruhr-Universität Bochum Medizinische Fakultät; F656-2009).
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Helling, S., Hüttemann, M., Kadenbach, B., Ramzan, R., Vogt, S., Marcus, K. (2012). Discovering the Phosphoproteome of the Hydrophobic Cytochrome c Oxidase Membrane Protein Complex. In: Marcus, K. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 893. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-885-6_21
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DOI: https://doi.org/10.1007/978-1-61779-885-6_21
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Publisher Name: Humana Press, Totowa, NJ
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