Although newly synthesized proteins play critical roles in cellular responses to foreign stimuli, only a small part of the total protein changes in response to an immunological stimulus ( e.g. less than 1% in stimulated RBL cells)(Chen, Centola et al. 1998). The majority of cellular responses are attributed to structural changes of proteins, rather than to the emergence of new proteins. Various forms of post-translational modifications (PTMs) such as phosphorylation, acetylation and lipidation have been identified. Protein phosphorylation is one of the most important types of PTM, playing a significant role in cell signalling and other biological activities of cell such as cell growth, division and metabolism (Graves and Krebs 1999). Reversible protein phosphorylation is a tuneable key for activating or inactivating many kinds of enzymes and receptors, the extent of phosphorylation, and therefore the cellular response, being regulated by the co-ordinated action of specific protein kinases and phosphatases. For this reason, regulatory proteins usually contain multiple phosphorylation sites (e.g. P53, (Milczarek, Martinez et al. 1996). In eukaryotes protein phosphorylation usually occurs on serine, threonine and tyrosine residues. A correlation between the increased number of tyrosine phosphorylation sites and species complexity, points to an important role for tyrosine-phosphate in evolution (Manning, Plowman et al. 2002).
In spite of the importance of protein phosphorylation in cell signalling, technical problems are still a major obstacle associated with the application of phosphoproteomics methods, which prohibits their use in daily routine experiments. One of the primary methods for phosphorylation identification is using radioactive phosphorous, but this method is not always a practical method and working with radioactive material is not desirable.
Recent developments in proteomics methods are applicable to phosphoproteomics investigations. In many cases general methods such as 2-D gel electrophoresis and Western blotting are very useful for phosphorylation detection and semi-quantitative analysis of phophoproteins. However, further investigation to identify the exact phosphorylation site usually requires complementary experiments with mass spectrometry (MS). For MS analysis, a primary step called “phosphoprotein/peptide enrichment” is essential. Recently, the tandem usage of enrichment methods with high throughput mass spectrometry has lead to promising development in phosphoproteomics (Zhou, Watts et al. 2001; Beausoleil, Jedrychowski et al. 2004; Brill, Salomon et al. 2004) .
Protein phosphorylation is a dynamic event. This means that the time point in cell activation is highly important and any delay to stop the activation process may lead to different results in phosphoprotein analysis. Additionally, about 30% of total intracellular proteins are phosphorylated at any given time, and therefore every potentially phosphorylable protein in live cell presents in the mixture of phosphorlylated and non-phosphorylated forms. Thus, even after identification of phosphoproteins, quantitative analysis is required to evaluate the change in the equilibrium between these two states. Furthermore, phosphorylated amino acids are relatively less abundant rather than non-phosphorylated amino acids among all protein sequence. Thus, when using peptides for identification appropriate phospho-peptide enrichment methods are required.
Three enrichment methods have been developed for phosphopetides; these are chemical modification, metal ion affinity based methods and immunoaffinity based methods.
Following phosphopeptide enrichment, sequencing methods with mass spectrometry and Edman degradation method are generally used for protein/peptide identification and phosphorylation map preparation.
Although many different techniques have been described in this field, there is no significant advantage for any individual method.
