53 research outputs found
A Bayesian semi-parametric model for thermal proteome profiling.
Funder: Wellcome TrustThe thermal stability of proteins can be altered when they interact with small molecules, other biomolecules or are subject to post-translation modifications. Thus monitoring the thermal stability of proteins under various cellular perturbations can provide insights into protein function, as well as potentially determine drug targets and off-targets. Thermal proteome profiling is a highly multiplexed mass-spectrommetry method for monitoring the melting behaviour of thousands of proteins in a single experiment. In essence, thermal proteome profiling assumes that proteins denature upon heating and hence become insoluble. Thus, by tracking the relative solubility of proteins at sequentially increasing temperatures, one can report on the thermal stability of a protein. Standard thermodynamics predicts a sigmoidal relationship between temperature and relative solubility and this is the basis of current robust statistical procedures. However, current methods do not model deviations from this behaviour and they do not quantify uncertainty in the melting profiles. To overcome these challenges, we propose the application of Bayesian functional data analysis tools which allow complex temperature-solubility behaviours. Our methods have improved sensitivity over the state-of-the art, identify new drug-protein associations and have less restrictive assumptions than current approaches. Our methods allows for comprehensive analysis of proteins that deviate from the predicted sigmoid behaviour and we uncover potentially biphasic phenomena with a series of published datasets
Hsp90 inhibition differentially destabilises MAP kinase and TGF-beta signalling components in cancer cells revealed by kinase-targeted chemoproteomics
<p>Abstract</p> <p>Background</p> <p>The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level.</p> <p>Methods</p> <p>We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ("kinobeads"). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure.</p> <p>Results</p> <p>We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain.</p> <p>Conclusions</p> <p>We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications.</p
Wilhelm et al. reply
REPLYING TO N. Fortelny, C. M. Overall, P. Pavlidis & G. V. Cohen Freue Nature 547, doi:10.1038/nature22293 (2017
Chemical proteomic analysis reveals the drugability of the kinome of Trypanosoma brucei
[Image: see text] The protozoan parasite Trypanosoma brucei is the causative agent of African sleeping sickness, and there is an urgent unmet need for improved treatments. Parasite protein kinases are attractive drug targets, provided that the host and parasite kinomes are sufficiently divergent to allow specific inhibition to be achieved. Current drug discovery efforts are hampered by the fact that comprehensive assay panels for parasite targets have not yet been developed. Here, we employ a kinase-focused chemoproteomics strategy that enables the simultaneous profiling of kinase inhibitor potencies against more than 50 endogenously expressed T. brucei kinases in parasite cell extracts. The data reveal that T. brucei kinases are sensitive to typical kinase inhibitors with nanomolar potency and demonstrate the potential for the development of species-specific inhibitors
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 (<sup>15</sup>N, <sup>13</sup>C) 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 <sup>15</sup>N- and <sup>13</sup>C-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, <sup>12</sup>C<sub>8</sub>H<sub>16</sub><sup>15</sup>N<sub>1</sub><sup>+</sup>; TMT127H, <sup>12</sup>C<sub>7</sub><sup>13</sup>C<sub>1</sub>H<sub>16</sub><sup>14</sup>N<sub>1</sub><sup>+</sup>; TMT129L, <sup>12</sup>C<sub>6</sub><sup>13</sup>C<sub>2</sub>H<sub>16</sub><sup>15</sup>N<sub>1</sub><sup>+</sup>; and TMT129H, <sup>12</sup>C<sub>5</sub><sup>13</sup>C<sub>3</sub>H<sub>16</sub><sup>14</sup>N<sub>1</sub><sup>+</sup>). 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 (<sup>15</sup>N, <sup>13</sup>C) 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 <sup>15</sup>N- and <sup>13</sup>C-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, <sup>12</sup>C<sub>8</sub>H<sub>16</sub><sup>15</sup>N<sub>1</sub><sup>+</sup>; TMT127H, <sup>12</sup>C<sub>7</sub><sup>13</sup>C<sub>1</sub>H<sub>16</sub><sup>14</sup>N<sub>1</sub><sup>+</sup>; TMT129L, <sup>12</sup>C<sub>6</sub><sup>13</sup>C<sub>2</sub>H<sub>16</sub><sup>15</sup>N<sub>1</sub><sup>+</sup>; and TMT129H, <sup>12</sup>C<sub>5</sub><sup>13</sup>C<sub>3</sub>H<sub>16</sub><sup>14</sup>N<sub>1</sub><sup>+</sup>). 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 (<sup>15</sup>N, <sup>13</sup>C) 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 <sup>15</sup>N- and <sup>13</sup>C-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, <sup>12</sup>C<sub>8</sub>H<sub>16</sub><sup>15</sup>N<sub>1</sub><sup>+</sup>; TMT127H, <sup>12</sup>C<sub>7</sub><sup>13</sup>C<sub>1</sub>H<sub>16</sub><sup>14</sup>N<sub>1</sub><sup>+</sup>; TMT129L, <sup>12</sup>C<sub>6</sub><sup>13</sup>C<sub>2</sub>H<sub>16</sub><sup>15</sup>N<sub>1</sub><sup>+</sup>; and TMT129H, <sup>12</sup>C<sub>5</sub><sup>13</sup>C<sub>3</sub>H<sub>16</sub><sup>14</sup>N<sub>1</sub><sup>+</sup>). 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
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