GW-5074 KiNativ -- dose response experiment - Dataset (ID:20088)
|HMS Dataset ID:||20088|
|Dataset Title:||GW-5074 KiNativ -- dose response experiment|
|Project Summary Page(s):||lincs.hms.harvard.edu/kinativ|
|Screening Lab Investigator:||Qingsong Liu|
|Screening Principal Investigator:||Nathanael Gray|
|Assay Description:||The KiNativ assay platform is based on the use of biotinylated acyl phosphates of ATP and ADP that act as probes by reacting with protein kinases on conserved lysine residues in the ATP binding pocket to covalently attach a biotin moiety. Using these probes, cell lysates treated with a kinase inhibitor can be labeled with BHAcATP or BHAcADP, with kinases inhibited by the compound of interest exhibiting reduced or no labeling. This is followed by digestion with trypsin, isolation of biotinylated peptides, and analysis by mass spectrometry to determine the extent of labeling of peptides from each kinase.|
1. Lyse cells in lysis buffer (20 mM HEPES, 150 mM NaCl, .1% Triton X-100, and 20 mM MnCl2).|
2. Centrifuge lysates at 100000 g for 60 min to remove insoluble matter.
3. Filter lysates using a .22 uM filter and gel filter into fresh lysis buffer using Bio-Rad 10DG columns to remove endogenous nucleotides (particularly ATP).
4. Adjust cell lysate to a total protein concentration of 5 mg/mL
5. Add kinase inhibitor to lysate for 5 min. Four different kinase inhibitor doses were tested for each compound. These final inhibitor concentration are 10 uM, 1 uM, 0.1 uM and 0.01 uM.
6. Label lysate with BHAcATP and BHAcADP at 20 uM for 5 min.
7. Reduce samples by treatment with DTT and block free cysteines with iodoacetamide, and gel filter to remove excess reagents.
9. Digest samples with trypsin.
10. Isolate probe-labeled peptides using streptavidin-agarose beads.
11. Wash beads and elute peptides with 50% CH3CN/water mixture with .1% TFA.
12. Analyze samples by LC-MS/MS: labeled kinase peptides will have a mass modified by 339 Da. Quantitate percent inhibition for each kinase. Data is presented as IC50 for assays using both the ADP and ATP probes.
13. For each compound, a total of 194 to 316 kinase labeling sites are monitored, depending on the individual experiment. Some kinases are labeled on multiple sites. For each labeling site, we report (1) the name/description of the kinase labeled, (2) the peptide sequence of the labeled binding site on the kinase, (3) the description of the labeling site (e.g. "located in activation loop"), (4) the derived IC50 for inhibition of substrate binding to the indicated site.
14. For some datasets there are labeling sites for which numeric data are not available. KiNativ has provided the following descriptions of these result value terms:
a. "ND": not determined, peptide was targeted but signal was insufficient for quantitation
b. "NI": no inhibition (< 35% change in MS signal in treated versus control sample)
c. "~NI": most likely not inhibited, in cases where MS signals were highly variable and/or weak
d. blank (null value): peptide not targeted
|Assay Protocol Reference:||
KiNativ website: kinativ.com |
Patricelli MP, Szardenings AK, Liyanage M, Nomanbhoy TK, Wu M, Weissig H, Aban A, Chun D, Tanner S, Kozarich JW. Functional interrogation of the kinome using nucleotide acyl phosphates. Biochemistry. 2007 Jan 16;46(2):350-8. PMID: 17209545
|HMS Dataset Type:||KiNativ|
|Date Publicly Available:||2011-07-15|
|Most Recent Update:||2016-09-09|