Identification of the Sites of Phosphorylation in Proteins Using High Performance Liquid Chromatography and Mass Spectrometry

Abstract

After incubation of cells with ³²P-labeled inorganic phosphate, it is possible to identify in vivo radiolabeled phosphoproteins. Generally, after the cells are lysed the phosphoprotein can be separated by sodium dodecyl sulfate (SDS) gel electrophoresis and a rough estimate of the size of the phosphoprotein can be gainnted. In order to determine the phosphorylated residues in the protein, the radiolabeled band can be transferred to a membrane, hydrolyzed with trypsin (or another suitable enzyme), and the two-dimensional (2D) map establishnted. The phosphopeptides observed on the 2D map can be tentatively correlated with expected tryptic fragments, based on their hydrophobicity and charge. A number of protocols have been developed to refine the correlation of the expected fragments to phosphopeptides present on the 2D map. For example, phospho-amino acid analysis of individual species present on the 2D map can be used to identify the type of phosphorylated residues present. In addition, manual Edman degradation can be performed on phosphopeptides after they are removed from the 2D map in order to identify the position of the ³²P-containing residue. This information can be used to help determine the position of the phosphorylated residue when more than one such residue is present in the tentatively assigned fragment sequence. Armed with this information, mutational analysis can confirm the site of phosphorylation, again using 2D map analysis. Although the above protocols have been successfully utilized, difficulties can arise. For example, the 2D map may not have sufficient resolution to separate two different phosphopeptides. As a result, the manual Edman analysis may not take into account the heterogeneous nature of the sample extractnted. Alternatively, ambiguous results can be obtained from manual Edmanwhen more than five cycles are required to discriminate between two possible “tentatively assigned” fragments. Partial oxidation of cysteine, methionine and tryptophan residue-containing peptides may also complicate the interpretation of the 2D map. Finally, the correlation between the pI and the hydrophobicity of the fragments may not be sufficient information to direct the mutational analysis. Many of these problems can be resolved by high-sensitivity mass spectrometry (MS) analysis. Analysis of peptide mixtures with MS will generally clarify whether a sample is heterogeneous and delineate modifications such as phosphorylation and oxidation.