Differential Kinobeads Profiling for Target Identification of Irreversible Kinase Inhibitors

Chemoproteomics profiling of kinase inhibitors with kinobeads enables the assessment of inhibitor potency and selectivity for endogenously expressed protein kinases in cell lines and tissues. Using a small panel of targeted covalent inhibitors, we demonstrate the importance of measuring covalent target binding in live cells. We present a differential kinobeads profiling strategy for covalent kinase inhibitors where a compound is added either to live cells or to a cell extract that enables the comprehensive assessment of inhibitor selectivity for covalent and noncovalent targets. We found that Acalabrutinib, CC-292, and Ibrutinib potently and covalently bind TEC family kinases, but only Ibrutinib also potently binds to BLK. ZAK was identified as a submicromolar affinity Ibrutinib off-target due to covalent modification of Cys22. In contrast to Ibrutinib, 5Z-7-Oxozeaenol reacted with Cys150 next to the DFG loop, demonstrating an alternative route to covalent inactivation of this kinase, e.g., to inhibit canonical TGF-β dependent processes

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

High-Resolution Enabled TMT 8‑plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation (¹⁵N, ¹³C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between ¹⁵N- and ¹³C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, ¹²C₈H₁₆¹⁵N₁⁺; TMT127H, ¹²C₇¹³C₁H₁₆¹⁴N₁⁺; TMT129L, ¹²C₆¹³C₂H₁₆¹⁵N₁⁺; and TMT129H, ¹²C₅¹³C₃H₁₆¹⁴N₁⁺). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays

Ion Coalescence of Neutron Encoded TMT 10-Plex Reporter Ions

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low mass range of tandem MS spectra for relative quantification. The recent extension of TMT multiplexing to 10 conditions has been enabled by utilizing neutron encoded tags with reporter ion <i>m</i>/<i>z</i> differences of 6 mDa. The baseline resolution of these closely spaced tags is possible due to the high resolving power of current day mass spectrometers. In this work we evaluated the performance of the TMT10 isobaric mass tags on the Q Exactive Orbitrap mass spectrometers for the first time and demonstrated comparable quantification accuracy and precision to what can be achieved on the Orbitrap Elite mass spectrometers. However, we discovered, upon analysis of complex proteomics samples on the Q Exactive Orbitrap mass spectrometers, that the proximate TMT10 reporter ion pairs become prone to coalescence. The fusion of the different reporter ion signals into a single measurable entity has a detrimental effect on peptide and protein quantification. We established that the main reason for coalescence is the commonly accepted maximum ion target for MS2 spectra of 1e6 on the Q Exactive instruments. The coalescence artifact was completely removed by lowering the maximum ion target for MS2 spectra from 1e6 to 2e5 without any losses in identification depth or quantification quality of proteins

A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level

Late stage failures of candidate drug molecules are frequently caused by off-target effects or inefficient target engagement <i>in vivo</i>. In order to address these fundamental challenges in drug discovery, we developed a modular probe strategy based on bioorthogonal chemistry that enables the attachment of multiple reporters to the same probe in cell extracts and live cells. In a systematic evaluation, we identified the inverse electron demand Diels–Alder reaction between <i>trans</i>-cyclooctene labeled probe molecules and tetrazine-tagged reporters to be the most efficient bioorthogonal reaction for this strategy. Bioorthogonal biotinylation of the probe allows the identification of drug targets in a chemoproteomics competition binding assay using quantitative mass spectrometry. Attachment of a fluorescent reporter enables monitoring of spatial localization of probes as well as drug-target colocalization studies. Finally, direct target occupancy of unlabeled drugs can be determined at single cell resolution by competitive binding with fluorescently labeled probe molecules. The feasibility of the modular probe strategy is demonstrated with noncovalent PARP inhibitors

A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level

Late stage failures of candidate drug molecules are frequently caused by off-target effects or inefficient target engagement <i>in vivo</i>. In order to address these fundamental challenges in drug discovery, we developed a modular probe strategy based on bioorthogonal chemistry that enables the attachment of multiple reporters to the same probe in cell extracts and live cells. In a systematic evaluation, we identified the inverse electron demand Diels–Alder reaction between <i>trans</i>-cyclooctene labeled probe molecules and tetrazine-tagged reporters to be the most efficient bioorthogonal reaction for this strategy. Bioorthogonal biotinylation of the probe allows the identification of drug targets in a chemoproteomics competition binding assay using quantitative mass spectrometry. Attachment of a fluorescent reporter enables monitoring of spatial localization of probes as well as drug-target colocalization studies. Finally, direct target occupancy of unlabeled drugs can be determined at single cell resolution by competitive binding with fluorescently labeled probe molecules. The feasibility of the modular probe strategy is demonstrated with noncovalent PARP inhibitors