Custom Mass Spectrometry Proteome Phosphorylation (MSPP) Services
Our Custom Mass Spectrometry Protein Phosphorylation (MSPP) Services provide for the detection of hundreds to thousands of phosphosites in biological specimens. These services use the sensitivity of a Thermo Electron LTQ Orbitrap tandem MS/MS spectrometer for global phosphorylation analyses. Several variations in methodology are available depending on the specific requirements of clients.
The most typical requests are for the identification of changes in protein phosphorylation in cultured cells in response to treatment with a drug or hormone, knockout of a gene or expression of mutated gene. For this purpose, Kinexus can culture control cells with normal isotopes to yield light phospho-peptides and in parallel cultivate treated cells with 3H4-Lysine- and 13C6-Arginine to produce heavy versions of the same phospho-peptides. Both groups of cells are harvested and lysed, heat and chaotropic agent-treated, and mixed. Following tryptic digestion, the mixed phosphopeptides are fractionated by isoelectric focusing and enriched by metal oxide chromatography with TiO2. Following further resolution and detection by LC-MS/MS, the phosphopeptides that demonstrate altered heavy:light ratio correlate to changes in protein phosphorylation: higher heavy:light ratios correspond to increased phosphorylation with the treatment. The identities of hundreds to thousands of phosphosites can be determined from the charge to mass ratio of the tryptic phospho-peptides when compared to the charge to mass ratios of all of the tryptic peptides from proteins encoded by the human genome and the genomes of other organisms that have been partially or fully sequenced.
The most typical requests are for the identification of changes in protein phosphorylation in cultured cells in response to treatment with a drug or hormone, knockout of a gene or expression of mutated gene. For this purpose, Kinexus can culture control cells with normal isotopes to yield light phospho-peptides and in parallel cultivate treated cells with 3H4-Lysine- and 13C6-Arginine to produce heavy versions of the same phospho-peptides. Both groups of cells are harvested and lysed, heat and chaotropic agent-treated, and mixed. Following tryptic digestion, the mixed phosphopeptides are fractionated by isoelectric focusing and enriched by metal oxide chromatography with TiO2. Following further resolution and detection by LC-MS/MS, the phosphopeptides that demonstrate altered heavy:light ratio correlate to changes in protein phosphorylation: higher heavy:light ratios correspond to increased phosphorylation with the treatment. The identities of hundreds to thousands of phosphosites can be determined from the charge to mass ratio of the tryptic phospho-peptides when compared to the charge to mass ratios of all of the tryptic peptides from proteins encoded by the human genome and the genomes of other organisms that have been partially or fully sequenced.
We can also provide phosphorylation analysis of cell/tissue lysates from patients and animal model experimental models by using post-homogenization labelling with heavy and light isotopes tags.
While other facilities may be capable of these mass spectrometry methods, Kinexus can provided deeper supplemental analyses for data interpretation. In conjunction with our PhosphoNET bioinformatics services, which include evolutionary and kinase prediction analyses, it is possible to distill out the most important changes in protein phosphorylation and interconnect them within signalling networks. Furthermore, Kinexus can assist in the development of phosphosite-specific antibodies for tracking these phosphorylation events in follow up studies.
These type of analyses can require hundreds of hours of instrumentation time and man hours of labour. Consequently, clients should consult with our Technical Services representatives about the scope of their intended projects so that realistic quotations can be provided. For studies involving the identification of thousands of phosphosites, the costs can be in the US$20,000 to US$30,000 range.
For more information on our Custom Mass Spectrometry Proteome Phosphorylation (MSPP) Services please download our Customer Information Package.
While other facilities may be capable of these mass spectrometry methods, Kinexus can provided deeper supplemental analyses for data interpretation. In conjunction with our PhosphoNET bioinformatics services, which include evolutionary and kinase prediction analyses, it is possible to distill out the most important changes in protein phosphorylation and interconnect them within signalling networks. Furthermore, Kinexus can assist in the development of phosphosite-specific antibodies for tracking these phosphorylation events in follow up studies.
These type of analyses can require hundreds of hours of instrumentation time and man hours of labour. Consequently, clients should consult with our Technical Services representatives about the scope of their intended projects so that realistic quotations can be provided. For studies involving the identification of thousands of phosphosites, the costs can be in the US$20,000 to US$30,000 range.
For more information on our Custom Mass Spectrometry Proteome Phosphorylation (MSPP) Services please download our Customer Information Package.
