Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP

Most kinase inhibitor drugs target the binding site of the nucleotide cosubstrate ATP. The high intracellular concentration of ATP can strongly affect inhibitor potency and selectivity depending on the affinity of the target kinase for ATP. Here we used a defined chemoproteomics system based on competition-binding assays in cell extracts from Jurkat and SK-MEL-28 cells with immobilized ATP mimetics (kinobeads). This system enabled us to assess the affinities of more than 200 kinases for the cellular nucleotide cofactors ATP, ADP, and GTP and the effects of the divalent metal ions Mg²⁺ and Mn²⁺. The affinity values determined in this system were largely consistent across the two cell lines, indicating no major dependence on kinase expression levels. Kinase-ATP affinities range from low micromolar to millimolar, which has profound consequences for the prediction of cellular effects from inhibitor selectivity profiles. Only a small number of kinases including CK2, MEK, and BRAF exhibited affinity for GTP. This extensive and consistent data set of kinase-nucleotide affinities, determined for native enzymes under defined experimental conditions, will represent a useful resource for kinase drug discovery

Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP

Most kinase inhibitor drugs target the binding site of the nucleotide cosubstrate ATP. The high intracellular concentration of ATP can strongly affect inhibitor potency and selectivity depending on the affinity of the target kinase for ATP. Here we used a defined chemoproteomics system based on competition-binding assays in cell extracts from Jurkat and SK-MEL-28 cells with immobilized ATP mimetics (kinobeads). This system enabled us to assess the affinities of more than 200 kinases for the cellular nucleotide cofactors ATP, ADP, and GTP and the effects of the divalent metal ions Mg²⁺ and Mn²⁺. The affinity values determined in this system were largely consistent across the two cell lines, indicating no major dependence on kinase expression levels. Kinase-ATP affinities range from low micromolar to millimolar, which has profound consequences for the prediction of cellular effects from inhibitor selectivity profiles. Only a small number of kinases including CK2, MEK, and BRAF exhibited affinity for GTP. This extensive and consistent data set of kinase-nucleotide affinities, determined for native enzymes under defined experimental conditions, will represent a useful resource for kinase drug discovery

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

Chemoproteomics Reveals Time-Dependent Binding of Histone Deacetylase Inhibitors to Endogenous Repressor Complexes

Class I histone deacetylases (HDACs) are attractive drug targets in oncology and inflammation. However, the development of selective inhibitors is complicated by the characteristic that the localization, activity, and selectivity of class I HDACs are regulated by association in megadalton repressor complexes. There is emerging evidence that isoform and protein complex selectivity can be achieved by aminobenzamide inhibitors. Here we present a chemoproteomics strategy for the determination of time-dependent inhibitor binding to endogenous HDACs and HDAC complexes. This approach enabled us to determine kinetic association and dissociation rates for endogenously expressed repressor complexes. We found that unlike hydroxamate type inhibitors, aminobenzamides exhibited slow binding kinetics dependent on association within protein complexes. These findings were in agreement with a delayed cellular response on acetylation levels of distinct histone sites and the inability of aminobenzamides to inhibit HDAC activity of a Sin3 complex isolated from K562 cells

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

Measuring and Managing Ratio Compression for Accurate iTRAQ/TMT Quantification

Isobaric mass tagging (e.g., TMT and iTRAQ) is a precise and sensitive multiplexed peptide/protein quantification technique in mass spectrometry. However, accurate quantification of complex proteomic samples is impaired by cofragmentation of peptides, leading to systematic underestimation of quantitative ratios. Label-free quantification strategies do not suffer from such an accuracy bias but cannot be multiplexed and are less precise. Here, we compared protein quantification results obtained with these methods for a chemoproteomic competition binding experiment and evaluated the utility of measures of spectrum purity in survey spectra for estimating the impact of cofragmentation on measured TMT-ratios. While applying stringent interference filters enables substantially more accurate TMT quantification, this came at the expense of 30%–60% fewer proteins quantified. We devised an algorithm that corrects experimental TMT ratios on the basis of determined peptide interference levels. The quantification accuracy achieved with this correction was comparable to that obtained with stringent spectrum filters but limited the loss in coverage to <10%. The generic applicability of the fold change correction algorithm was further demonstrated by spiking of chemoproteomics samples into excess amounts of <i>E. coli</i> tryptic digests