Mass Spectrometry Services
Kinexus is pleased to offer four proteomics services for protein and phosphorylation site identification. These further augment the powerful suite of unique and integrated proteomics methodologies offered by Kinexus for biomarker discovery.
Our Protein Identification by Mass Spectrometry (PIMS) Services are designed to permit our clients to establish the identities of cross-immunoreactive proteins that demonstrate interesting changes in expression or phosphorylation in their experimental model systems. Such proteins are visualized with our Kinetworks™ multi-immunoblotting services and may also be detected with our Kinase-Substrate Profiling Services. The PIMS service requires that the target cross-reactive protein can be immunoprecipitated or supplied in a purified form ready for trypsinolysis and resolution by LC-MS/MS mass spectrometry. Since the apparent molecular mass of the antibody cross-reactive target protein is known, this can be used to further validate the identification of the protein from the estimated charge to mass ratios of its tryptic fragments. Such protein fingerprinting is a very powerful and sensitive strategy to identify proteins using our knowledge of the predicted charge to mass ratios of all of the proteins encoded by the genomes of humans and other species that have been sequenced.
Our Custom Mass Spectrometry Proteome Phosphorylation (MSPP) Services provide for the detection of hundreds to thousands of phosphosites that are present in cell lysates, and which may be subject to perturbation in experimental model systems. With this method, lysate phosphoproteins are currently trypsinized and the phosphorylated fragments fractionated by isoelectrofocusing and enriched by titanium oxide purification prior to resolution by LC tandem MS/MS mass spectrometry. In conjunction with our PhosphoNET bioinformatics services, it is possible to distill out the most important changes in protein phosphorylation and interconnect them within signalling networks.
Our Custom Mass Spectrometry Proteome Phosphorylation (MSPP) Services provide for the detection of hundreds to thousands of phosphosites that are present in cell lysates, and which may be subject to perturbation in experimental model systems. With this method, lysate phosphoproteins are currently trypsinized and the phosphorylated fragments fractionated by isoelectrofocusing and enriched by titanium oxide purification prior to resolution by LC tandem MS/MS mass spectrometry. In conjunction with our PhosphoNET bioinformatics services, 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.
Our Custom Mass Spectrometry Protein Phosphosite (MSPS) Profiling Services provide for the detection of multiple phosphosites that are present in a purified phosphoprotein. The phosphoprotein may be isolated by immunoprecipitation from cells treated in vivo or it could be a recombinant protein that is phosphorylated by a protein kinase in vitro. With this method, the phosphoprotein is trypsinized and the phosphorylated fragments enriched by titanium oxide purification prior to resolution by LC tandem MS/MS mass spectrometry.
Our Custom Mass Spectrometry Kinase-Substrate (MSKS) Profiling Services allow the identification of physiological substrates and their phosphosites that are targeted by a purified protein kinase that is added to a cell lysate. The cells must be separately cultivated in medium that contains amino acids that are labeled with either light and heavy isotopes. With this method, after in vitro kinase phosphorylation of the heavy isotope-labeled proteins, the lysates from the separately cultured cells are mixed. Subsequently, the phosphoproteins are trypsinized, and the phosphorylated fragments enriched by titanium oxide purification prior to resolution by LC tandem MS/MS mass spectrometry. An selective increase in phosphorylation of the heavy isotope-labeled phosphopeptides provides for the detection of likely substrates for the added protein kinase. This method not only identifies physiological substrates, but the alignments of the targeted phosphosites can reveal information about the substrate specificity of the added kinase.
Our Custom Mass Spectrometry Protein Phosphosite (MSPS) Profiling Services provide for the detection of multiple phosphosites that are present in a purified phosphoprotein. The phosphoprotein may be isolated by immunoprecipitation from cells treated in vivo or it could be a recombinant protein that is phosphorylated by a protein kinase in vitro. With this method, the phosphoprotein is trypsinized and the phosphorylated fragments enriched by titanium oxide purification prior to resolution by LC tandem MS/MS mass spectrometry.
Our Custom Mass Spectrometry Kinase-Substrate (MSKS) Profiling Services allow the identification of physiological substrates and their phosphosites that are targeted by a purified protein kinase that is added to a cell lysate. The cells must be separately cultivated in medium that contains amino acids that are labeled with either light and heavy isotopes. With this method, after in vitro kinase phosphorylation of the heavy isotope-labeled proteins, the lysates from the separately cultured cells are mixed. Subsequently, the phosphoproteins are trypsinized, and the phosphorylated fragments enriched by titanium oxide purification prior to resolution by LC tandem MS/MS mass spectrometry. An selective increase in phosphorylation of the heavy isotope-labeled phosphopeptides provides for the detection of likely substrates for the added protein kinase. This method not only identifies physiological substrates, but the alignments of the targeted phosphosites can reveal information about the substrate specificity of the added kinase.
For more information on our Custom Mass Spectrometry Protein Phosphosite (MSPS) Profiling Services and our Custom Mass Spectrometry Kinase-Substrate (MSKS) Profiling Services please contact our Technican Services representatives toll free at 1-866-KINEXUS or by e-mail at info@kinexus.ca.