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Dataset Information
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Dataset Name:
CH5424802 KiNativ -- single dose experiment

Dataset 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.

--Data in Package:
20105.txt

--Metadata in Package:
Small_Molecule_Metadata.txt
Protein_Metadata.txt
Cell_Line_Metadata.txt

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Center-specific Information
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Center-specific Name:
HMS_LINCS

Center-specific Dataset ID:
20105

Center-specific Dataset Link:
http://lincs.hms.harvard.edu/db/datasets/20105/

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Assay Information
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Assay Protocol:
1.  Lyse cells in lysis buffer ( 20 mM HEPES, 150 mM NaCl, .1% Triton X-100, and 20 mM MnCl2).<br />
2.  Centrifuge lysates at 100000 g for 60 min to remove insoluble matter.<br />
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).<br />
4.  Adjust cell lysate to a total protein concentration of 5 mg/mL.<br />
5.  Add kinase inhibitor to lysate for 5 min. One kinase inhibitor dose was tested for each compound. The final inhibitor concentration is 10 uM.<br />
6.  Label lysate with BHAcATP and BHAcADP at 20 uM for 5 min.<br />
7.  Reduce samples by treatment with DTT and block free cysteines with iodoacetamide, and gel filter to remove excess reagents.<br />
9.  Digest samples with trypsin.<br />
10.  Isolate probe-labeled peptides using streptavidin-agarose beads.<br />
11.  Wash beads and elute peptides with 50% CH3CN/water mixture with .1% TFA.<br />
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 % inhibition by compound for assays using both the ADP and ATP probes.<br />
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 percent inhibition of substrate binding to the indicated site.<br />
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:<br />
a. "ND": not determined, peptide was targeted but signal was insufficient for quantitation<br />
b. "NI": no inhibition (< 35% change in MS signal in treated versus control sample)<br />
c. "~NI": most likely not inhibited, in cases where MS signals were highly variable and/or weak<br />
d. blank (null value): peptide not targeted<br />

Date Updated:
2016-09-09

Date Retrieved from Center:
11/13/2015

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Metadata Information
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Metadata information regarding the entities used in the experiments is included in the accompanied metadata. A metadata file per entity category is included in the package. For example, the metadata for all the cell lines that were used in the dataset are included in the Cell_Lines_Metadata.txt file.
Descriptions for each metadata field can be found here: http://www.lincsproject.org/data/data-standards/
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