Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/ProteinQuantitaion_GlobalProteomics.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/20250620_Michele_Kinativ_Report.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HEP_G2_DMSO_1uL_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HT1080_DMSO_1uL_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HT1080_LATSi782_1uL_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/KRASi_NCGC1607_1uM_6uL_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/KRASi_NCGC1607_1uM_6uL_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/KRASi_noCpd_6uL_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/DMSO_NCGC1607_1uM_6uL_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HEP_G2_LATSi782_1uL_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HEP_G2_DMSO_1uL_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/KRASi_NCGC1607_100nM_6uL_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/DMSO_noCpd_6uL_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HT1080_LATSi782_1uL_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/DMSO_noCpd_6uL_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/KRASi_NCGC1607_100nM_6uL_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HEP_G2_LATSi782_1uL_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HT1080_DMSO_1uL_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HEP_G2_DMSO_1uL_R3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HEP_G2_LATSi782_1uL_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/KRASi_noCpd_6uL_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HT1080_LATSi782_1uL_R4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HT1080_LATSi782_1uL_R1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/KRASi_NCGC1607_100nM_6uL_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/DMSO_noCpd_6uL_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HT1080_DMSO_1uL_R4_20250213133325.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/DMSO_NCGC1607_100nM_6uL_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HT1080_DMSO_1uL_R2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HEP_G2_DMSO_1uL_R4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/DMSO_NCGC1607_100nM_6uL_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/DMSO_NCGC1607_1uM_6uL_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/KRASi_noCpd_6uL_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/KRASi_NCGC1607_1uM_6uL_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/HEP_G2_LATSi782_1uL_R4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/DMSO_NCGC1607_1uM_6uL_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD073488/DMSO_NCGC1607_100nM_6uL_2.rawprimaryOK200dingyin.tao@nih.govDingyin TaoMass SpectrometryBottom-up proteomicsData-independent acquisitionHumanProteomicsKinase inhibitorKinase selectivity profilinghttps://www.ebi.ac.uk/pride/archive/projects/PXD073488Cell CultureLysates for label-free global proteomic profiling were prepared from HEP-G2 and HT-1080 cells following 24 hours of treatment with either NCGC-6782 (1 µM) or DMSO (0.1%). The cells were washed with ice-cold PBS supplemented with protease inhibitor and phosphatase inhibitor cocktails (Sigma), detached using a cell scraper, and pelleted at 8,000 rpm for 5 min. at 4°C. Total proteins were extracted from the cell pellets using ice-cold Pierce RIPA buffer (Thermo Fisher) supplemented with protease inhibitor and phosphatase inhibitor cocktails (Sigma). Cells were incubated in RIPA buffer for 15 minutes on ice, followed by sonication (10 sec on, 15 sec off, for a total time of 2 minutes at 50% power). After sonication, the lysates were cleared by centrifugation at 13,000 rpm for 10 min. at 4°C and protein yields were quantified using Pierce BCA protein assay kit (Thermo Fisher). Protein concentrations were normalized based on BCA results and samples were frozen at -80°C until analysis. The cell lysates were denatured and reduced with the addition of 0.1% RapidGest (Waters Corporation) and 15 mM TCEP at 55°C for 45 min, and alkylated with 15 mM IAA in the dark for 30 min. The alkylated proteins were then precipitated by adding 9:1 (v/v, ACN/Protein sample) of cold ACN (-20 ºC) and incubating for 15 min on ice. After aspirating the supernatant, the proteins were redissolved in trypsin buffer (25 mM NH4 hCO3, pH 8.0) at an enzyme/substrate ratio (W/W) of 1:50 and incubated at 37°C overnight. The digestion was quenched by adding 1% of FA (final concentration), and the supernatant was transferred to a clean and protein low-bind tube for further LC-MS/MS analysis. Cellular based kinome profilings were performed using the Pierce™ Kinase Enrichment Kit with ActivX™ ATP Probe (Thermo Fisher). Briefly, 200 MLNs Hek293LX cells were lysed in Pierce IP Lysis Buffer (supplemented with protease-inhibitors and phosphatase-inhibitors cocktails) using Dounce homogenizer. After 15’ incubation in ice, the lysate was clarified by centrifugation, and the resulting supernatant was desalted with Zeba Spin Desalting Column. Following BCA quantification, the lysate was diluted to 2 mg/mL with Reaction buffer. Probe labeling and active site peptide enrichment were performed using Pierce™ Kinase Enrichment Kits with ActivX™ Probes (Thermo scientific) according to the manufacturer’s recommendation, with minor modification. Briefly, 500 μL lysate aliquots (2 mg/mL) were sequentially incubated with 20 mM MgCl2 for 1 minute, DMSO (Millipore Sigma) or kinase inhibitors for 10 minutes, and desthiobiotin-ATP probe for 10 minutes at room temperature. The probe-labeled lysates were then denatured and reduced with the addition of 4.9 M Urea/IP Lysis Buffer and 4.8 mM Tris(2-carboxyethyl)phosphine (TCEP, Millipore Sigma) at 55°C for 45 min, alkylated with 37 mM iodoacetamide (IAA, Millipore Sigma) in the dark for 30 min at room temperature, and buffer-exchanged into 2 M Urea/20 mM ammonium bicarbonate (NH4 hCO3, Millipore Sigma, pH 8.0) using Zeba Spin Desalting Column. After desalting, the lysate proteins were digested with Trypsin/Lys-C (Promega) at an enzyme/substrate ratio (W/W) of 1:50 at 37°C overnight. The desthiobiotinylated peptides were then captured using Streptavidin Agarose resins. To remove non-specific binding, the resins were extensively washed with Pierce IP Lysis Buffer, 1×PBS (Gibco), and MS-grade water (Millipore Sigma). Finally, the labeled peptides were eluted from the resins using 50% acetonitrile (ACN, Thermo Fisher) with 0.1% trifluoroacetic acid (TFA, Millipore Sigma) and stored at -20°C after lyophilization.PrideNot availableProteome Discoverer software (version 3.1, Thermo Fisher) was used for peptide identification and quantitation. Searches were performed using CHIMERYS v2.7.17 with inferys 3.0.0 fragmentation prediction model. The MS raw data were searched against a Swiss-Prot human database (version Nov 2022, reviewed database) consisting of 20,328 entries using the following parameters: Max. fragment mass tolerance was set as 20 ppm; up to two miss cleavages was set for trypsin digestion; fixed modification considered was carbamidomethylation of cysteine; variable modification considered was oxidation of methionine residues. Peptide identification false discovery rates (FDR) were limited to a maximum of 0.01. Label-free quantification analysis used the “Fragment Ions Quantifier” node from Proteome Discoverer The raw data was searched by Spectronaut 18.6 in directDIA47 mode using a FASTA database created from all known human proteins from Swissprot (downloaded Feb 2024), with the following parameters: precursor q-value cutoff of 0.01, precursor PEP cutoff of 0.2, protein q-value cutoff (experiment) of 0.01, protein q-value cutoff (run) of 0.05, and protein PEP cutoff of 0.75. Decoy sequences were generated with the ‘mutated’ method and with preferred fragment source NN predicted fragments, with library size fraction of 0.1. Post-translational modifications were analyzed with PTM localization probability cutoff of 0.75 and PTM consolidation using sum. Cross-run normalization was performed with filter type ‘keep if any’ for Biotin:Thermo-88310 using local regression normalization. The remainder of the search settings were used with BGS factory settings. For kinome analysis, further quality control and comparative analysis was performed in R 4.4.0 using the following packages: tidyverse (version 2.0.0) for data formatting and preprocessing, ensembldb (version 2.28.0), EnsDb.Hsapiens.v86 (version 2.99.0), and biomaRt (version 2.60.0) for annotation, and cowplot (version 1.1.3), VennDiagram (version 1.7.3), RColorBrewer (version 1.1-3), pheatmap (1.01.12) and ggExtra (version 0.10.1) for plotting. To process the data, all peptides were exported from Spectronaut and then loaded into an R dataframe using read_tsv, and all non-biotinylated peptides were filtered out by searching for the modification Biotin:Thermo-88310, then all unlocalized biotinylation sites were also filtered. To determine the position of lysine 1 and lysine 2, the sequence surrounding the biotin modification was extracted from the peptide using stringr and known lysine 1 and lysine 2 motifs were matched using regexes35. To remove all non-kinase hits from the kinome data, a list of 636 known human kinases were downloaded from the Uniprot/Swissprot database using biomaRt (keyword KW-0418, taxonomy_id:9606, downloaded 2023.01.24)ProteomicsDingyin TaoPARTIALNational Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, USA.Homo Sapiens (human)dingyin.tao@nih.gov41699947 Tharakan R, Qu Y, Ceribelli M, Morris PJ, Fang Y, Thomas CJ, LeClair CA, Tao D. Data-Independent Acquisition Enhancement of a Competitive Activity-Based Protein Profiling Platform for Kinase Inhibitor Screening. J Mass Spectrom. 2026 61(3):e70038 10.1002/jms.70038National Institutes of Health (NIH) National Center for Advancing Translational Sciences (NCATS)United StatesKinase inhibitors represent a vital class of therapeutic agents widely used in cancer research, immunology, and other disease areas. Mass spectrometry (MS) employing specially designed small-molecule kinase-binding probes has become an essential strategy for identifying novel kinase drug targets. While traditional MS approaches often rely on targeted proteomics (e.g., multiple reaction monitoring [MRM]) or data-dependent acquisition (DDA), data-independent acquisition (DIA) offers broader and more reproducible quantification, especially for low-abundance peptides. In this study, we systematically developed an activity-based protein profiling (ABPP) platform leveraging DIA, through integrated in-house informatics tools for data filtering and motif analysis, to provide an effective kinase profiling workflow. Compared to DDA, the DIA approach yielded more than a 100% increase in identified biotinylated peptides and over 40% improvement in kinase peptide coverage, while reducing the analysis time by half (90 min vs. 180 min per sample). Additionally, there was a modest improvement to the coefficient of variation (CV) in kinase peptide quantification (decrease from 11.41% to 10.70%; mean CV). Shorter liquid chromatography (LC) gradient times (60, 45, and 30 min) were evaluated as a means for increasing sample analysis throughput. Notably, no significant loss in kinase peptide coverage was observed due to shorter gradients, highlighting the capability of DIA to significantly enhance the efficiency and scalability of kinase profiling workflows.Data-Independent Acquisition Enhancement of a Competitive Activity-Based Protein Profiling Platform for Kinase Inhibitor Screening.Tharakan Ravi R, Qu Yanyan Y, Ceribelli Michele M, Morris Patrick J PJ, Fang Yuhong Y, Thomas Craig J CJ, LeClair Christopher A CA, Tao Dingyin DfalseKinome-profiling enabled discovery of a LATS1/2 inhibitor with wound healing potentialSystem-level mapping of kinase inhibitors polypharmacology can uncover opportunities for drug repurposing and identify starting points for medicinal chemistry campaigns aimed at novel targets. Here, we describe a large-scale profiling of 257 kinase inhibitors that revealed domains of promiscuity, pockets of selectivity and a broad region of uninhibited targets. Focusing on a subcluster enriched for AGC group kinases and using the AKT inhibitor AT-7867 (an antiproliferative agent) as a starting point, we employed scaffold hopping and property-based optimization to discover NCGC-6782, a LATS1/2 inhibitor with robust pro-proliferative activity. In cells, NCGC-6782 inhibited LATS1/2 activity, promoted rapid YAP nuclear translocation and transactivation of YAP-target genes. Kinome-wide profilings demonstrated good in vitro selectivity and exquisite chemoproteomic and kinetic selectivity. Transcriptomics and proteomics following NCGC-6782 revealed upregulation of YAP-dependent genes (4h) followed by activation of cell-cycle programs (24h). Consistently, NCGC-6782 accelerated proliferative regeneration in both cell-based and in vivo wound healing 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