title: Improvement of capture compound mass spectrometry technology (CCMS) for the profiling of human kinases by combination with 2D LC-MS/MS.
abstract: An increasingly popular and promising field in functional proteomics is the isolation of proteome subsets based on small molecule-protein interactions. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate captured protein conjugates from complex biological samples for direct protein identification by liquid chromatography/mass spectrometry (nLC-MS/MS). In this study we used staurosporine as a selectivity group for analysis in HepG2 cells derived from human liver. In the present study, we combined the functional isolation of kinases with different separation workflows of automated split-free nanoflow liquid chromatography prior to mass spectrometric analysis. Two different CCMS setups, CCMS technology combined with 1D LC-MS and 2D LC-MS, were compared regarding the total number of kinase identifications. By extending the chromatographic separation of the tryptic digested captured proteins from 1D LC linear gradients to 2D LC we were able to identify 97 kinases. This result is similar to the 1D LC setup we previously reported but this time 4 times less input material was needed. This makes CCMS of kinases an even more powerful tool for the proteomic profiling of this important protein family.
authors.affiliation: Analytics Department, caprotec bioanalytics GmbH, Volmerstrasse 5, 12489 Berlin, Germany.
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Fischer
authors.forename: Olivia
authors.initials: O
authors.lastname: Graebner
authors.forename: Mathias
authors.initials: M
authors.lastname: Dreger
authors.forename: Mirko
authors.initials: M
authors.lastname: Glinski
authors.forename: Sabine
authors.initials: S
authors.lastname: Baumgart
authors.forename: Hubert
authors.initials: H
authors.lastname: Koester
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D056945:Hep G2 Cells; D006801:Humans; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D010770:Phosphotransferases / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D019311:Staurosporine / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D010770:Phosphotransferases; D019311:Staurosporine