Kinase chromatography and mass spectrometry was performed as described previously (33 (link)). Briefly, compounds were commercially obtained or synthesized directly, and then affixed to sepharose using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-catalyzed peptide coupling chemistry. Cell lysates were then diluted in binding buffer with 1M NaCl and affinity purification was performed with gravity chromatography. The bound kinases were stringently washed and then eluted with hot SDS before extraction and tryptic digest. Liquid chromatography-tandem mass spectrometry (LC MS/MS) was performed on a Velos Orbitrap (Thermo) with in-line HPLC using an EASY-spray column (Thermo). Label-free quantification was performed with Skyline (44 (link)), and statistical analysis with Ms Stats (45 (link)).
Protocol detail
Kinase Affinity Profiling by Chromatography and Mass Spectrometry
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Affinity purification
Buffer
Carbodiimide
Cell
Chromatography
Gravity
Hplc
Kinases
Liquid chromatography
Mass spectrometry
Nacl
Peptide
Sepharose
Tandem mass spectrometry
Tryptic
Corresponding Organization : Harvard University
Other organizations : University of California, San Francisco, UCSF Helen Diller Family Comprehensive Cancer Center, Center for Cancer Research, Wellcome Sanger Institute, Lahey Medical Center, Lahey Hospital and Medical Center, University of Rochester, Sapporo Higashi Tokushukai Hospital, Howard Hughes Medical Institute
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Variable analysis
independent variables
- Compounds commercially obtained or synthesized directly
- Kinase binding and elution
- Protein identification and quantification by LC-MS/MS
- 1M NaCl in binding buffer
- Stringent washing of bound kinases
- Hot SDS elution of bound kinases
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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