Protein phosphorylation studying

Detection of phosphorylated proteins and peptides can be enhanced by selectively isolating these species. Online immobilized metal affinity chromatography (IMAC)-CE-ESI-MS is such a powerful analytical tool. The IMAC resin retains and preconcentrates phosphorylated proteins and peptides, CE separates the phosphorylated species and MS/MS identifies the components and their phosphorylation sites. Cao and Stults applied this method to the analysis of phosphorylated angiotensin II and tryptic digests of a- and /3-casein (CE conditions buffer, 0.1% acetic acid/10% methanol uncoated capillary). Beta-casein is a well-characterized protein with five phosphorylation sites and is widely used as a standard for protein phosphorylation studies. 

Calmodulin, a calcium binding protein, is involved in Ca2+-dependent regulation of several synaptic functions of the brain synthesis, uptake and release of neurotransmitters, protein phosphorylation and Ca+2 transport. It reacts with TET, TMT and TBT which then inactivates enzymes like Ca+2-ATPase and phosphodiesterase. In vitro studies indicated TBT was greater at inhibiting calmodulin activity than TET and TMT, whereas in vivo the order was TET > TMT > TBT. This may be due to the greater detoxification of TBT (66%) in the liver before moving to other organs30,31. 

The study of protein phosphorylation has helped to clarify the mechanisms involved in the causes and manifestation of disorders of the nervous system. Two illustrative examples are given here Alzheimer s disease and opiate addiction. 

Other PTMs may involve changes in the chemical nature of amino acids (e.g., citrullination or deimination). Because many of these modifications result in mass changes that are measurable by MS, they are amenable to detection by MS-based approaches. A number of emerging MS-based strategies allow the identification of PTMs. Several MS-based methods to determine the types and sites of protein phosphorylation and ubiquitination have been developed. Phosphorylation occurs mainly on serine, threonine, and tyrosine residues at a frequency ratio of 1800 200 1 in vertebrates.70 Although the phosphorylation of tyrosine residues occurs less frequently in the proteome, it has been extensively studied. 

Src is the prototype of the superfamily of protein tyrosine kinases and was one of the first protein kinases to be characterized by various genetic, cellular, and structure-function studies to help imderstand its role in signal transduction pathways as well as in disease processes, including cancer, osteoporosis, and both tumor- and inflammation-mediated bone loss [28-38]. In fact, studies on Src provided some of the first evidence correlating protein kinase activity and substrate protein phosphorylation in the regulation of signal transduction pathways relative to normal cellular activity as well as mahgnant transformations. Src family kinases include Fyn, Yes, Yrk, Blk, Fgr, Hck, Lyn,... 

Our results demonstrated that the identified subsets of the activated protein kinases significantly increased the accuracy of clinical outcome predictions. Most notably in the study, we evaluated protein phosphorylation levels instead of total protein expression levels. Protein phosphorylation and dephosphorylation are well-characterized biochemical processes for protein kinases to conduct cellular signal transduction. Phosphorylation at certain tyrosine, serine, or threonine residues in kinases is a key step for their activation, and the measurement of these phosphorylations reflects their functional status in vivo. Thus, the protein kinase phosphorylation-based tissue microarray more accurately reveals the molecular mechanisms of breast cancers, and more accurately predicts the individualized survival and treatment response. 

With respect to sample preparation, it is necessary to develop effective and fast procedures involving only a few steps in order to avoid contamination, reduce analysis time and to improve the quality of analytical work. Microsampling and the use of smaller sample sizes is required and also the further development of analytical techniques. In particular, there is a need for the development of online and/or hyphenated techniques in ICP-MS. Microsampling combined with the separation of small amounts of analytes will be relevant for several chromatographic techniques (such as the development of micro- and nano-HPLC). There is a demand for further development of the combination of LA-ICP-MS as an element analytical technique with a biomolecular mass spectrometric technique such as MALDI- or ESI-MS for molecular identification and quantification of protein phosphorylation as well as of metal concentrations, this also enables the study of post-translational modifications of proteins, e.g. phosphorylation. 

Barford, D. (1999) Structural studies of reversible protein phosphorylation and protein phosphatases. Biochem. Soc. Trans. 27, 751-766. 

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