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High-throughput Identification of FLT3 Wild-type and Mutant Kinase Substrate Preferences and Application to Design of Sensitive In Vitro Kinase Assay Substrates

2018; Elsevier BV; Volume: 18; Issue: 3 Linguagem: Inglês

10.1074/mcp.ra118.001111

1535-9484

Minervo Perez, John F. Blankenhorn, Kevin Murray, Laurie L. Parker,

Cancer-related Molecular Pathways

Acute myeloid leukemia (AML) is an aggressive disease that is characterized by abnormal increase of immature myeloblasts in blood and bone marrow. The FLT3 receptor tyrosine kinase plays an integral role in hematopoiesis, and one third of AML diagnoses exhibit gain-of-function mutations in FLT3, with the juxtamembrane domain internal tandem duplication (ITD) and the kinase domain D835Y variants observed most frequently. Few FLT3 substrates or phosphorylation sites are known, which limits insight into FLT3's substrate preferences and makes assay design particularly challenging. We applied in vitro phosphorylation of a cell lysate digest (adaptation of the Kinase Assay Linked with Phosphoproteomics (KALIP) technique and similar methods) for high-throughput identification of substrates for three FLT3 variants (wild-type, ITD mutant, and D835Y mutant). Incorporation of identified substrate sequences as input into the KINATEST-ID substrate preference analysis and assay development pipeline facilitated the design of several peptide substrates that are phosphorylated efficiently by all three FLT3 kinase variants. These substrates could be used in assays to identify new FLT3 inhibitors that overcome resistant mutations to improve FLT3-positive AML treatment. Acute myeloid leukemia (AML) is an aggressive disease that is characterized by abnormal increase of immature myeloblasts in blood and bone marrow. The FLT3 receptor tyrosine kinase plays an integral role in hematopoiesis, and one third of AML diagnoses exhibit gain-of-function mutations in FLT3, with the juxtamembrane domain internal tandem duplication (ITD) and the kinase domain D835Y variants observed most frequently. Few FLT3 substrates or phosphorylation sites are known, which limits insight into FLT3's substrate preferences and makes assay design particularly challenging. We applied in vitro phosphorylation of a cell lysate digest (adaptation of the Kinase Assay Linked with Phosphoproteomics (KALIP) technique and similar methods) for high-throughput identification of substrates for three FLT3 variants (wild-type, ITD mutant, and D835Y mutant). Incorporation of identified substrate sequences as input into the KINATEST-ID substrate preference analysis and assay development pipeline facilitated the design of several peptide substrates that are phosphorylated efficiently by all three FLT3 kinase variants. These substrates could be used in assays to identify new FLT3 inhibitors that overcome resistant mutations to improve FLT3-positive AML treatment. Acute myeloid leukemia (AML) 1The abbreviations used are:AMLacute myeloid leukemiaRTKreceptor tyrosine kinaseITDinternal tandem duplicationTKDtyrosine kinase domainWTwild typeTKItyrosine kinase inhibitorKALIPKinase Assay Linked PhosphoproteomicsFBSfetal bovine serumPBSphosphate buffered salineACNacetonitrileDTTdithiothreitolTFAtrifluoroacetic acidHLBhydrophilic-lipophilic balancedTris-HCLtris(hydroxymethyl)aminomethane hydrochlorideEDTAethylenediaminetetraacetic acidFAformic acidFDRfalse discovery rateMCCMatthews Correlation CoefficientAAamino acidDMFdimethylformamideDMSOdimethyl sulfoxidePDGFRβplatelet derived growth factor betaALKanaplastic leukemia kinaseBTKbruton's tyrosine kinaseMOPS4-morpholinepropanesulfonic acidBSAbovine serum albuminHEPES4-(2-hydroxyethyl)-1-piperzoneethanesulfonic acidTBStris buffered salineARamplex red2Htwo hours16H16 hoursSDVstandard deviation valuesPPMpositional probability matrixSSMsite selectivity matrixDRdose response. 1The abbreviations used are:AMLacute myeloid leukemiaRTKreceptor tyrosine kinaseITDinternal tandem duplicationTKDtyrosine kinase domainWTwild typeTKItyrosine kinase inhibitorKALIPKinase Assay Linked PhosphoproteomicsFBSfetal bovine serumPBSphosphate buffered salineACNacetonitrileDTTdithiothreitolTFAtrifluoroacetic acidHLBhydrophilic-lipophilic balancedTris-HCLtris(hydroxymethyl)aminomethane hydrochlorideEDTAethylenediaminetetraacetic acidFAformic acidFDRfalse discovery rateMCCMatthews Correlation CoefficientAAamino acidDMFdimethylformamideDMSOdimethyl sulfoxidePDGFRβplatelet derived growth factor betaALKanaplastic leukemia kinaseBTKbruton's tyrosine kinaseMOPS4-morpholinepropanesulfonic acidBSAbovine serum albuminHEPES4-(2-hydroxyethyl)-1-piperzoneethanesulfonic acidTBStris buffered salineARamplex red2Htwo hours16H16 hoursSDVstandard deviation valuesPPMpositional probability matrixSSMsite selectivity matrixDRdose response. is an aggressive cancer with a diverse genetic landscape. The FLT3 gene encodes for a receptor tyrosine kinase (FLT3) that regulates hematopoiesis and perturbations to its signaling pathways appear to promote AML disease progression. In fact, FLT3 is implicated as a major factor in AML relapse (1Leick M.B. Levis M.J. The Future of Targeting FLT3 Activation in AML.Curr. Hematol. Malig. Rep. 2017; 12: 153-167Crossref PubMed Scopus (35) Google Scholar). Thirty percent of AML cases have mutations to FLT3 that lead the kinase to be constitutively active (2Stirewalt D.L. Radich J.P. The role of FLT3 in haematopoietic malignancies.Nat. Rev. Cancer. 2003; 3: 65-650Crossref PubMed Scopus (706) Google Scholar, 3Pozarowski P. Darzynkiewicz Z. Analysis of cell cycle by flow cytometry.Methods Mol. Biol. 2004; 281: 11-301Google Scholar) most commonly to the juxtamembrane domain and the kinase domain (2Stirewalt D.L. Radich J.P. The role of FLT3 in haematopoietic malignancies.Nat. Rev. Cancer. 2003; 3: 65-650Crossref PubMed Scopus (706) Google Scholar, 4Leung A. Man C.H. Kwong Y.-L. FLT3 inhibition: a moving and evolving target in acute myeloid leukemia.Nat. Rev. Leuk. 2013; 27: 260-268Google Scholar, 5Yamamoto Y. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies.Blood. 2001; 97: 2434-2439Crossref PubMed Scopus (974) Google Scholar). Internal tandem duplication (FLT3-ITD) in the juxtamembrane or the first tyrosine kinase domain (TKD) occurs when a segment is duplicated (head to tail) leading to the loss of repressive regions in the protein (6Yoshimoto G. Miyamoto T. Jabbarzadeh-Tabrizi S. Iino T. Rocnik J.L. Kikushige Y. Mori Y. Shima T. Iwasaki H. Takenaka K. Nagafuji K. Mizuno S. Niiro H. Gilliland G.D. Akashi K. FLT3-ITD up-regulates MCL-1 to promote survival of stem cells in acute myeloid leukemia via FLT3-ITD – specific STAT5 activation.Blood. 2009; 114: 5034-5044Crossref PubMed Scopus (173) Google Scholar). A second common mutation is a substitution of aspartic acid 835 to a tyrosine residue (D835Y) in the TKD. Both ITD and TKD mutants can activate and dimerize with the wild type FLT3 (7Kiyoi H. Ohno R. Ueda R. Saito H. Naoe T. Mechanism of constitutive activation of FLT3 with internal tandem duplication in the juxtamembrane domain.Oncogene. 2002; 21: 2555-2563Crossref PubMed Scopus (237) Google Scholar). The effects of these mutations on FLT3 signaling are still unclear, but one possibility is that mutant FLT3-TKD and FLT3-ITD activate alternative signaling pathways, or activate standard FLT3 pathways aberrantly, compared with the WT. Mutations to FLT3 are correlated with poor long-term prognosis (8Swords R. Freeman C. Giles F. Targeting the FMS-like tyrosine kinase 3 in acute myeloid leukemia.Leukemia. 2012; 26: 2176-2185Crossref PubMed Scopus (107) Google Scholar, 9Kim Y. Lee G.D. Park J. Yoon J.H. Kim H.J. Min W.S. Kim M. Quantitative fragment analysis of FLT3 -ITD ef fi ciently identifying poor prognostic group with high mutant allele burden or long ITD length.Nature. 2015; 5: e336-e377Google Scholar) and although patients with FLT3 mutations achieve similar initial disease remission to those with wild-type FLT3, they have an increased risk for relapse (2Stirewalt D.L. Radich J.P. The role of FLT3 in haematopoietic malignancies.Nat. Rev. Cancer. 2003; 3: 65-650Crossref PubMed Scopus (706) Google Scholar, 8Swords R. Freeman C. Giles F. Targeting the FMS-like tyrosine kinase 3 in acute myeloid leukemia.Leukemia. 2012; 26: 2176-2185Crossref PubMed Scopus (107) Google Scholar, 10Smith C.C. Lin K. Stecula A. Sali A. Shah N.P. FLT3 D835 mutations confer differential resistance to type II FLT3 inhibitors.Leukemia. 2015; 29: 2390-2392Crossref PubMed Scopus (122) Google Scholar). In vitro studies show that FLT3-ITD mutant-expressing cell lines are resistant to cytosine arabinoside (the primary AML therapeutic) (8Swords R. Freeman C. Giles F. Targeting the FMS-like tyrosine kinase 3 in acute myeloid leukemia.Leukemia. 2012; 26: 2176-2185Crossref PubMed Scopus (107) Google Scholar). These findings prompted the use of a combinatorial approach to AML therapies to include FLT3 tyrosine kinase inhibitors (TKIs), which are frequently initially successful but often lead to FLT3 inhibitor resistance and subsequent disease relapse. acute myeloid leukemia receptor tyrosine kinase internal tandem duplication tyrosine kinase domain wild type tyrosine kinase inhibitor Kinase Assay Linked Phosphoproteomics fetal bovine serum phosphate buffered saline acetonitrile dithiothreitol trifluoroacetic acid hydrophilic-lipophilic balanced tris(hydroxymethyl)aminomethane hydrochloride ethylenediaminetetraacetic acid formic acid false discovery rate Matthews Correlation Coefficient amino acid dimethylformamide dimethyl sulfoxide platelet derived growth factor beta anaplastic leukemia kinase bruton's tyrosine kinase 4-morpholinepropanesulfonic acid bovine serum albumin 4-(2-hydroxyethyl)-1-piperzoneethanesulfonic acid tris buffered saline amplex red two hours 16 hours standard deviation values positional probability matrix site selectivity matrix dose response. acute myeloid leukemia receptor tyrosine kinase internal tandem duplication tyrosine kinase domain wild type tyrosine kinase inhibitor Kinase Assay Linked Phosphoproteomics fetal bovine serum phosphate buffered saline acetonitrile dithiothreitol trifluoroacetic acid hydrophilic-lipophilic balanced tris(hydroxymethyl)aminomethane hydrochloride ethylenediaminetetraacetic acid formic acid false discovery rate Matthews Correlation Coefficient amino acid dimethylformamide dimethyl sulfoxide platelet derived growth factor beta anaplastic leukemia kinase bruton's tyrosine kinase 4-morpholinepropanesulfonic acid bovine serum albumin 4-(2-hydroxyethyl)-1-piperzoneethanesulfonic acid tris buffered saline amplex red two hours 16 hours standard deviation values positional probability matrix site selectivity matrix dose response. The current FDA approved TKIs used to inhibit FLT3 were not developed specifically to target FLT3 (11Moore a S. Faisal A. Gonzalez de Castro D. Bavetsias V. Sun C. Atrash B. Valenti M. de Haven Brandon A. Avery S. Mair D. Mirabella F. Swansbury J. Pearson A.D. Workman P. Blagg J. Raynaud F.I. Eccles S.A. Linardopoulos S. Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns.Leukemia. 2012; 26: 1462-1470Crossref PubMed Scopus (94) Google Scholar, 12Grunwald M.R. Levis M.J. FLT3 inhibitors for acute myeloid leukemia: a review of their efficacy and mechanisms of resistance.Int. J. Hematol. 2013; 97: 94-683Crossref Scopus (129) Google Scholar, 13Daver N. Cortes J. Ravandi F. Patel K.P. Burger J.A. Konopleva M. Kantarjian H. Secondary mutations as mediators of resistance to targeted therapy in leukemia.Blood. 2015; 125: 10-20Crossref PubMed Scopus (95) Google Scholar). Sorafenib is a type II pan-TKI which is FDA approved for use in combinatorial approaches with AML chemotherapy, but elicits no response in FLT3 variants with tyrosine kinase domain mutations (8Swords R. Freeman C. Giles F. Targeting the FMS-like tyrosine kinase 3 in acute myeloid leukemia.Leukemia. 2012; 26: 2176-2185Crossref PubMed Scopus (107) Google Scholar, 14Metzelder S.K. Schroeder T. Finck A. Scholl S. Fey M. Götze K. Linn Y.C. Kröger M. Reiter A. Salih H.R. Heinicke T. Stuhlmann R. Müller L. Giagounidis A. Meyer R.G. Brugger W. Vöhringer M. Dreger P. Mori M. Basara N. Schäfer-Eckart K. Schultheis B. Baldus C. Neubauer A. Burchert A. High activity of sorafenib in FLT3-ITD-positive acute myeloid leukemia synergizes with allo-immune effects to induce sustained responses.Leukemia. 2012; 26: 2353-2359Crossref PubMed Scopus (174) Google Scholar, 15Lindblad O. Cordero E. Puissant A. Macaulay L. Ramos A. Kabir N.N. Sun J. Vallon-Christersson J. Haraldsson K. Hemann M.T. Borg Å. Levander F. Stegmaier K. Pietras K. Rönnstrand L. Kazi J.U. Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML.Oncogene. 2016; 35: 5119-5131Crossref PubMed Scopus (76) Google Scholar, 16Weisberg E. Sattler M. Ray A. Griffin J.D. Drug resistance in mutant FLT3-positive AML.Oncogene. 2010; 29: 5120-5134Crossref PubMed Scopus (80) Google Scholar, 17Renneville A. Roumier C. Biggio V. Nibourel O. Boissel N. Fenaux P. Preudhomme C. Cooperating gene mutations in acute myeloid leukemia: a review of the literature.Leukemia. 2008; 22: 915-931Crossref PubMed Scopus (294) Google Scholar). Efforts to develop FLT3 mutant-specific TKIs lead to the discovery of the type II TKI quizartinib, which can inhibit the FLT3-ITD mutant and is currently undergoing phase III clinical trials for AML (18Larrosa-Garcia M. Baer M.R. FLT3 Inhibitors in Acute Myeloid Leukemia: Current Status and Future Directions.Mol. Cancer Ther. 2017; 16: 991-1001Crossref PubMed Scopus (185) Google Scholar). However, quizartinib has no activity against FLT3-TKD point mutations and thus these mutations are the primary mode of quizartinib monotherapy resistance (18Larrosa-Garcia M. Baer M.R. FLT3 Inhibitors in Acute Myeloid Leukemia: Current Status and Future Directions.Mol. Cancer Ther. 2017; 16: 991-1001Crossref PubMed Scopus (185) Google Scholar, 19Wander Sa. Levis M.J. Fathi A.T. The evolving role of FLT3 inhibitors in acute myeloid leukemia: quizartinib and beyond.Ther. Adv. Hematol. 2014; 5: 65-77Crossref PubMed Scopus (143) Google Scholar, 20Williams A.B. Nguyen B. Li L. Brown P. Levis M. Leahy D. Small D. Mutations of FLT3/ITD confer resistance to multiple tyrosine kinase inhibitors.Leukemia. 2013; 27: 48-55Crossref PubMed Scopus (70) Google Scholar, 21Zarrinkar P.P. Gunawardane R.N. Cramer M.D. Gardner M.F. Brigham D. Belli B. Karaman M.W. Pratz K.W. Pallares G. Chao Q. Sprankle K.G. Patel H.K. Levis M. Armstrong R.C. James J. Bhagwat S.S. AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML).Blood. 2009; 114: 2984-2992Crossref PubMed Scopus (462) Google Scholar). Quizartinib also has potent activity toward Platelet Derived Growth Factor receptor (PDGFR) and c-KIT kinases, and produces side effects that may be related to their inhibition in patients undergoing a FLT3 TKI regimen (22Galanis A. Ma H. Rajkhowa T. Ramachandran A. Small D. Cortes J. Levis M. Crenolanib is a potent inhibitor of flt3 with activity against resistance-Conferring point mutants.Blood. 2014; 123: 94-100Crossref PubMed Scopus (181) Google Scholar, 23Cortes, J. E., Kantarjian, H., Foran, J. M., Ghirdaladze, D., Zodelava, M., Borthakur, G., Gammon, G., Trone, D., Armstrong, R. C., James, J., and Levis, M., Phase IStudy of Quizartinib administered daily to patients with relapsed or refractory acute myeloid leukemia irrespective of FMS-like tyrosine kinase 3–internal tandem duplication status. Clin. J. Oncol. 31, 3681–3687.Google Scholar). Crenolanib, a TKI designed to target the α and β isoforms of PDGFR, has demonstrated activity against a broad range of FLT3 mutations (1Leick M.B. Levis M.J. The Future of Targeting FLT3 Activation in AML.Curr. Hematol. Malig. Rep. 2017; 12: 153-167Crossref PubMed Scopus (35) Google Scholar, 24Zimmerman E.I. Turner D.C. Buaboonnam J. Hu S. Orwick S. Roberts M.S. Janke L.J. Ramachandran A. Stewart C.F. Inaba H. Baker S.D. Crenolanib is active against models of drug-resistant FLT3-ITD 2 positive acute myeloid leukemia.Myeloid Neoplasia. 2013; 122: 3607-3615Google Scholar). Unlike quizartinib, crenolanib does not inhibit c-KIT (the main kinase implicated in undesirable side effects of quizartinib) at safe plasma concentrations, and is undergoing phase II clinical trials in relapsed AML patients with a driver FLT3 mutation (NCT01657682) (22Galanis A. Ma H. Rajkhowa T. Ramachandran A. Small D. Cortes J. Levis M. Crenolanib is a potent inhibitor of flt3 with activity against resistance-Conferring point mutants.Blood. 2014; 123: 94-100Crossref PubMed Scopus (181) Google Scholar, 25Smith C.C. Lasater E.A. Lin K.C. Wang Q. McCreery M.Q. Stewart W.K. Damon L.E. Perl A.E. Jeschke G.R. Sugita M. Carroll M. Kogan S.C. Kuriyan J. Shah N.P. Crenolanib is a selective type I pan-FLT3 inhibitor.Proc Natl Acad Sci. 2014; 111: 5319-5324Crossref PubMed Scopus (140) Google Scholar). However, recent reports have shown that secondary point mutations within the kinase domain of FLT3 can reduce crenolanib's clinical efficacy that suggest it is only a matter of time until crenolanib resistant mutations are found in a clinical setting (22Galanis A. Ma H. Rajkhowa T. Ramachandran A. Small D. Cortes J. Levis M. Crenolanib is a potent inhibitor of flt3 with activity against resistance-Conferring point mutants.Blood. 2014; 123: 94-100Crossref PubMed Scopus (181) Google Scholar, 25Smith C.C. Lasater E.A. Lin K.C. Wang Q. McCreery M.Q. Stewart W.K. Damon L.E. Perl A.E. Jeschke G.R. Sugita M. Carroll M. Kogan S.C. Kuriyan J. Shah N.P. Crenolanib is a selective type I pan-FLT3 inhibitor.Proc Natl Acad Sci. 2014; 111: 5319-5324Crossref PubMed Scopus (140) Google Scholar). The complex abnormality landscape of AML reduces the possibility that a single FLT3 TKI would be a viable monotherapy for AML. Although crenolanib is a promising TKI, efficient development of new inhibitors will require better assays than those currently available, and adaptable strategies that effectively screen inhibitors to target mutant forms of FLT3 are especially needed (18Larrosa-Garcia M. Baer M.R. FLT3 Inhibitors in Acute Myeloid Leukemia: Current Status and Future Directions.Mol. Cancer Ther. 2017; 16: 991-1001Crossref PubMed Scopus (185) Google Scholar). Because very little is known about FLT3 substrate preferences, there are few options available when designing FLT3 activity assays. The current activity tests are limited by inefficient phosphorylation activity, and/or their phosphorylation by the mutant variants has not been characterized. In this manuscript, we describe the development of several novel and efficient peptide substrates for FLT3 and two clinically-significant mutant variants (the ITD and D835Y mutants). We adapted the "Kinase Assay Linked with Phosphoproteomics" (KALIP) (26Xue, L., Wang, W. H., Iliuk, A., Hu, L., Galan, J. A., Yu, S., Hans, M., Geahlen, R. L., and Tao, W. A., Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates. Proc Natl Acad Sci. 109, 5615–5620.Google Scholar, 27Iliuk A.B. Martin Va Alicie B.M. Geahlen R.L. Tao W.A. In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers.Mol. Cell. Proteomics. 2010; 9: 2162-2172Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) strategy (from the Tao lab) to perform high-throughput determination of FLT3's preferred peptide substrate motif in a manner similar to other previously reported methods (e.g. Kettenbach et al. from the Gerber group) (28Kettenbach A.N. Wang T. Faherty B.K. Madden D.R. Knapp S. Bailey-Kellogg C. Gerber S.A. Rapid determination of multiple linear kinase substrate motifs by mass spectrometry.Chem Biol. 2012; 19: 608-618Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In these approaches, a cell lysate digest is stripped of endogenous phosphorylation and used in a kinase reaction as a pseudo-"library" of peptides to determine kinase substrate preferences by identifying phosphorylated sequences via enrichment and mass spectrometry (ideal for high-throughput analysis of many substrates simultaneously without requiring radioactivity or other labeling) (29Meyer N.O. O'Donoghue A.J. Schulze-Gahmen U. Ravalin M. Moss S.M. Winter M.B. Knudsen G.M. Craik C.S. Multiplex substrate profiling by mass spectrometry for kinases as a method for revealing quantitative substrate motifs.Anal Chem. 2017; 89: 4550-4558Crossref PubMed Scopus (12) Google Scholar, 30Lubner J.M. Balsbaugh J.L. Church G.M. Chou M.F. Schwartz D. Characterizing Protein kinase substrate specificity using the proteomic peptide library (ProPeL) approach.Curr. Protoc. Chem. Biol. 2018; 10: e38Crossref PubMed Scopus (7) Google Scholar). We then used the identified substrate preferences to rationally design a panel of candidate peptides incorporating key sequence features predicted to make them favorable for phosphorylation by the FLT3 kinase variants, following our previously reported substrate development pipeline KINATEST-ID (31Lipchik A.M. Perez M. Bolton S. Dumrongprechachan V. Ouellette S.B. Cui W. Parker L.L. KINATEST-ID: A pipeline to develop phosphorylation-dependent terbium sensitizing kinase assays.Am. J. Chem. Soc. 2015; 137: 2484-2494Crossref PubMed Scopus (25) Google Scholar). We demonstrated that these substrates enable efficient inhibitor screening for all three forms of FLT3. These peptides could be used in many different types of drug discovery settings to more rapidly and efficiently screen for and validate FLT3 inhibition. KG-1 cells (ATCC) were maintained in IMDM media (Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin in 5% CO2 at 37 °C. KG-1 cells were washed with 30 mLs of phosphate buffered saline (PBS) 5 times. The cells were then pelleted at 1,500 RPM for 5 min and lysed with buffer containing 8 m urea, 0.1 m ammonium bicarbonate pH 8.5, 20% acetonitrile (ACN), 20 mm dithiothreitol (DTT), and 1× Pierce Phosphatase Inhibitor tablet (Roche) pH 8.0. Lysed cells were incubated on ice for 15 min and then were subjected to probe sonication to shear the DNA. Lysates were treated with 40 mm iodoacetamide and incubated at room temperature (protected from light) for 60 min. Samples were then centrifuged at 15,000 RPM for 30 min to remove cellular debris. Urea concentration was diluted to 1.5 m using 50 mm ammonium bicarbonate buffer (pH 8.0) and the samples were set up for trypsin digestion at a 1:50 trypsin (ThermoScientific) ratio and incubated at 37 °C overnight. Trypsin digestion was quenched by adding 10% trifluoroacetic acid (TFA) in water to lower the pH below 3. Subsequently, the tryptic digest was desalted using hydrophilic-lipophilic balanced copolymer (HLB) reverse phase cartridges (Waters) and vacuum dried. Samples were reconstituted in alkaline phosphatase dephosphorylation buffer containing 50 mm tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCL), 0.1 mm Ethylenediaminetetraacetic acid (EDTA) at pH 8.5. Alkaline phosphatase (6 U, Roche) were added to each sample followed by incubation for 90 min at 37 °C. The reaction was quenched by incubating the samples in 75 °C for 15 min (Fig. 1). Recombinant kinases were purchased from EMD Millipore (WT, PN: PV3182; FLT3-D835Y, PN: PV3967; FLT3-ITD, PN: PV6190). The samples were briefly vortexed and aliquoted into two equal parts. Peptide samples were reconstituted in kinase reaction buffer containing 50 mm Tris HCL, pH 7.5, 10 mm MgCl2, 1 mm DTT, 1 mm Na3VO4 and 2 mm adenosine 5′-triphosphate (ATP). The kinase (or water for control) was added last to each sample and incubated for 16 h (16 H) at 37 °C. The FLT3-WT treatment contained an additional two-hour (2H) time point. The reaction was quenched by bringing up the concentration of TFA to 0.5% and desalted using Oasis HLB 1cc cartridge columns (Waters) with 30-micron particle size and 30 milligram sorbent. The phosphopeptide enrichments were carried out according to manufacturer's instructions (Tymora Analytical, West Lafayette, IN) (26Xue, L., Wang, W. H., Iliuk, A., Hu, L., Galan, J. A., Yu, S., Hans, M., Geahlen, R. L., and Tao, W. A., Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates. Proc Natl Acad Sci. 109, 5615–5620.Google Scholar, 27Iliuk A.B. Martin Va Alicie B.M. Geahlen R.L. Tao W.A. In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers.Mol. Cell. Proteomics. 2010; 9: 2162-2172Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The enrichment kit is made up of four components: (1) loading buffer, (2) PolyMAC magnetic beads, (3) wash buffer 1 and 2, and (4) elution buffer. In brief, the dried peptides were re-suspended in Loading Buffer and 100 μl of PolyMAC capture beads were added to the mixture. The phosphopeptide-PolyMAC mixture was mixed at 700 RPM for 30 min. Subsequently, the mixture was centrifuged briefly and placed on a magnetic rack to remove the un-phosphorylated peptide solution. The beads were washed twice with wash buffer 1 and rocked for 5 min at 700 RPM. The phosphopeptide-PolyMAC complex was placed on the magnetic stand until beads were immobilized by the magnet, and the supernatant was discarded; the process was repeated using wash buffer 2. The phosphopeptides were eluted from the capture beads using 300 μl of elution buffer and then vacuum dried. Samples were reconstituted in 25 μl of mass spectrometry loading buffer (98/2/0.5%; H2O/ACN/formic acid (FA)) and centrifuged for 30 min at 15,000 RPM. A 20 μl aliquot was transferred to a low binding safe-lock microtube (Eppendorf). A 2.5 μl aliquot was loaded on a ThermoScientific Easy NanoLC LC 1000 system. The reverse phased HPLC peptide separation was performed using a 100 μm inner diameter Picotip emitter column packed in-house with 1.9 μm C18 ReproSil-Pur sorbent. The mobile phase consisted of 0.1% formic acid in ultra-pure water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B). Samples were run over a linear gradient (2–30% solvent B; 60 min) with a flow rate of 200 nL/min into a high resolution Orbitrap Fusion Tribrid Mass Spectrometer, operated using data dependent mode at a resolution of 60,000 with a scan range of 300–1500 m/z. After each round of precursor detection, an MS/MS experiment was triggered on the top 12 most abundant ions using High Collision Dissociation (HCD). The mass analyzer parameters were set between two and seven charge states with a dynamic exclusion time of 15 s. The Orbitrap Fusion mass spectra files were searched against a merged version of the reviewed human Uniprot database downloaded from uniprot.org (2/27/2017; 20,202 entries) and the cRAP database (common lab contaminants; downloaded from (thegpm.org/crap/) on 2/27/2017) using the Paragon algorithm in the ProteinPilot 5.0 proteomic search engine within the Galaxy-P pipeline to create a Distinct Peptide Report (the output report from ProteinPilot 5.0) (32Afgan E. Baker D. van den Beek M. Blankenberg D. Bouvier D. Èech M. Chilton J. Clements D. Coraor N. Eberhard C. Grüning B. Guerler A. Hillman-Jackson J. Von Kuster G. Rasche E. Soranzo N. Turaga N. Taylor J. Nekrutenko A. Goecks J. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update.Nucleic Acids Res. 2016; 44: W3-W10Crossref PubMed Scopus (1246) Google Scholar, 33Boekel J. Kumar P. Easterly C. Esler M. Mehta S. Eschenlauer A.C. Hegeman A.D. Jagtap P.D. Griffin T.J. Multi-omic data analysis using Galaxy.Nat Biotechnol. 2015; 33: 137-139Crossref PubMed Scopus (98) Google Scholar, 34Shilov I.V. Seymour S.L. Patel A.A. Loboda A. Tang W.H. Keating S.P. Hunter C.L. Nuwaysir L.M. Schaeffer D.A. The paragon algorithm a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.Mol Cell Proteomics. 2007; 6: 1638-1655Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar). Peptide precursor mass tolerance was set at 0.02 Da and MS/MS tolerance was set to 0.1 Da. Proteomic database search parameters included trypsin digestion, urea denaturation, phosphorylation emphasis, iodoacetamide fixed modification to cysteine residues, and variable biological modifications. False discovery rate (FDR) analysis was activated for each individual search. ProteinPilot 5.0 used a reverse database as the decoy to calculate the false discovery rate (FDR) for each independent search (35Tang W.H. Shilov I.V. Seymour S.L. Nonlinear fitting method for determining local false discovery rates from decoy database searches.J. Proteome Res. 2008; 7: 3661-3667Crossref PubMed Scopus (274) Google Scholar) and we set the global 1% FDR score as our cutoff threshold. A series of novel scripts were developed to prepare and analyze the results from KALIP to design potential substrates in the KINATEST-ID platform. These steps and scripts are described in more detail in the supplemental methods, and detailed instructions on running each script in sequence are provided in the supporting information file "kinatestsop.docx." To extract and reformat the phosphopeptide sequences from the ProteinPilot distinct peptide report, we created the KinaMine program and GUI that extracts all sequences from a ProteinPilot 5.0 (SCIEX) Distinct Peptides Report output file that have phosphorylated tyrosine residues identified at a 99% confidence (1% FDR), and creates "Substrate" and "Substrate Background Frequency (SBF)" files, which contain the observed substrate sequences and the UniProt (uniprot.org) accession numbers and calculated representation of all amino acids for the proteins from which substrate sequences were identified, respectively. We created the "commonality and difference finder.r" script to identify the phosphopeptides from the "substrates" and SBF files that are shared by all of the FLT3 kinase variants, and generated the "SHARED-16H" substrate and SBF files. We ext