Tyrosine phosphorylation, technique

A number of techniques have been applied to profile tyrosine phosphorylation status (10) [reviewed in (4)]. Most existing approaches are based on a mass-spectrometry (MS) detection system, which is low-throughput, time-consuming, and labor intensive [reviewed in (4)]. Recently, the antibody array has been used for profiling the expression of tyrosine phosphorylated proteins. However, it does not provide information on other important aspects of tyrosine phosphorylation, such as pathway interaction partners or associated signaling pathways. 

Post-source Decay Tandem Mass Spectrometry The potential of PSD has also been exploited to identify phosphopeptides [61,62]. This technique is used for MALDI-generated ions in a reflectron TOP instrument. PSD can also distinguish any serine-threonine phosphorylation from tyrosine phosphorylation. Phosphotyrosine-containing peptides mostly yield the [MH — HP03J+ (loss of 80 Da) ion, whereas phosphoserine- and threonine-containing peptides produce [MH - H3P041+ (loss of 98 Da) and [M - HP03]+ ions. 

The use of QM-MD as opposed to QM-MM minimization techniques is computationally intensive and thus precluded the use of an ab initio or density functional method for the quantum region. This study was performed with an AMi Hamiltonian, and the first step of the dephosphorylation reaction was studied (see Fig. 4). Because of the important role that phosphorus has in biological systems [62], phosphatase reactions have been studied extensively [63]. From experimental data it is believed that Cys-i2 and Asp-i29 residues are involved in the first step of the dephosphorylation reaction of BPTP [64,65]. Alaliambra et al. [30] included the side chains of the phosphorylated tyrosine, Cys-i2, and Asp-i 29 in the quantum region, with link atoms used at the quantum/classical boundaries. In this study the protein was not truncated and was surrounded with a 24 A radius sphere of water molecules. Stochastic boundary methods were applied [66]. 

Phosphorylation on serine, threonine, and tyrosine residues is an extremely important modulator of protein function. Phosphorylation can be analyzed by mass spectrometry with enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopep-tides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags (McLachlin and Chait 2001). 

Other oncogenes were soon discovered in a variety of animal viruses, and as the techniques of DNA sequencing became more routine, a surprising number of these oncogenes were matched up with normal genes in animals and even in humans. But what was the role of these proto-oncogenes Most of them have now been shown to code for enzymes that help control the growth and differentiation of cells. In many instances, the enzymes are kinases that phosphorylate serine, threonine or tyrosine residues of other... 

Many ancillary techniques have been developed in recent years, but several stand out in their ability to extend the usefulness of 2D6EL. This final section will discuss four of these Western blotting, antibody production from gel spots, and metabolic labelling to determine amino acid composition and tyrosine-specific phosphorylation. 

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