Mass Spectrometry Kinase-Substrate (MSKS) Profiling Services
Kinexus is pleased to offer a powerful mass spectrometry-based approach to identify potential physiological substrates in cell lysates for protein kinases of high interest. This methodology does not require any knowledge about the specificity of a target kinase or the availability of phosphosite antibodies that cross-react with the kinase’s substrates. It can potentially identify hundreds of substrate phosphosites for a target kinase.
Clients can supply their own preparations of active protein kinases or choose from over 340 different protein kinases available through Kinexus. Because the method relies on the use of Stable Isotope Labeling of cultured Cells (SILAC), it is recommended that clients use one of the 16 human cell lines commonly grown by Kinexus or provide the desired cells to Kinexus for propagation and SILAC treatment.
For preparation of the cell lysates, “control” cells are cultivated in the presence of amino acids with common isotopes, i.e. 12C-Arg and 1H-Lys, to yield “light” isotope labeled proteins. In parallel cultures, the same cell line is also grown in the presence of amino acids with rare isotopes, i.e. 13C-Arg and 2H-Lys, to yield “heavy” isotope labeled proteins and generate “treated” cells. Following the lysis of both populations of cells, the lysate proteins are treated with preparations of phosphatases to reduce background protein phosphorylation.
Clients can supply their own preparations of active protein kinases or choose from over 340 different protein kinases available through Kinexus. Because the method relies on the use of Stable Isotope Labeling of cultured Cells (SILAC), it is recommended that clients use one of the 16 human cell lines commonly grown by Kinexus or provide the desired cells to Kinexus for propagation and SILAC treatment.
For preparation of the cell lysates, “control” cells are cultivated in the presence of amino acids with common isotopes, i.e. 12C-Arg and 1H-Lys, to yield “light” isotope labeled proteins. In parallel cultures, the same cell line is also grown in the presence of amino acids with rare isotopes, i.e. 13C-Arg and 2H-Lys, to yield “heavy” isotope labeled proteins and generate “treated” cells. Following the lysis of both populations of cells, the lysate proteins are treated with preparations of phosphatases to reduce background protein phosphorylation.
Upon removal of the phosphatases, ATP is added to both the “light” and “heavy” isotope labeled lysate protein preparations, and a catalytically active preparation of the kinase of interest is also added only to the “heavy” isotope labeled lysate protein preparation. Following further incubation at room temperature for ~30 minutes, both lysates are heat treated in the presence of a chaotropic agent. The two lysate preparations are the mixed and incubated with trypsin to produce peptides. The phosphorylated peptides are fractionated by isoelectric focusing, enriched by TiO2 chromatography and then subjected to LC-MS/MS tandem mass spectrometry. Due to the differential labeling of the “light” and “heavy” phospho-peptides, they emerge as doublets in the MS traces. If the added kinase targets a phosphosite in the phospho-peptide, then the second peak of the doublet is much larger. From the charge to mass ratio of the phospho-peptide, it can be identified.
To obtain more information about our Custom Mass Spectrometry Kinase Substrate Identification (MSKS) Service please download our Customer Information Package for this service. After reviewing this package, interested clients should contact our Technical Services representatives toll free at 1-866-KINEXUS or by e-mail at info@kinexus.ca.
To obtain more information about our Custom Mass Spectrometry Kinase Substrate Identification (MSKS) Service please download our Customer Information Package for this service. After reviewing this package, interested clients should contact our Technical Services representatives toll free at 1-866-KINEXUS or by e-mail at info@kinexus.ca.
