RESUMO
Multiplexed quantitative proteomics enabled complex workflows to study the mechanisms by which small molecule drugs interact with the proteome such as thermal proteome profiling (TPP) or multiplexed proteome dynamics profiling (mPDP). TPP measures changes in protein thermal stability in response to drug treatment and thus informs on direct targets and downstream regulation events, while the mPDP approach enables the discovery of regulated protein synthesis and degradation events caused by small molecules and other perturbations. The isobaric mass tags available for multiplexed proteomics have thus far limited the efficiency and sensitivity by which such experiments could be performed. Here we evaluate a recent generation of 16-plex isobaric mass tags and demonstrate the sensitive and time efficient identification of Staurosporine targets in HepG2 cell extracts by recording full thermal denaturation/aggregation profiles of vehicle and compound treated samples in a single mass spectrometry experiment. In 2D-TPP experiments, isothermal titration over seven concentrations per temperature enabled comprehensive selectivity profiling of Staurosporine with EC50 values for kinase targets tightly matching to the kinobeads gold standard assay. Finally, we demonstrate time and condition-based multiplexing of dynamic SILAC labeling experiments to delineate proteome-wide effects of the molecular glue Indisulam on synthesis and degradation rates.
Assuntos
Preparações Farmacêuticas , Proteômica , Espectrometria de Massas , Estabilidade Proteica , ProteomaRESUMO
CRISPR/Cas9-based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40-300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , Inativação Gênica/fisiologia , Células Hep G2 , Humanos , Mutação INDEL/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
We describe a two-dimensional thermal proteome profiling strategy that can be combined with an orthogonal chemoproteomics approach to enable comprehensive target profiling of the marketed histone deacetylase inhibitor panobinostat. The N-hydroxycinnamide moiety is identified as critical for potent and tetrahydrobiopterin-competitive inhibition of phenylalanine hydroxylase leading to increases in phenylalanine and decreases in tyrosine levels. These findings provide a rationale for adverse clinical observations and suggest repurposing of the drug for treatment of tyrosinemia.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Fenilalanina Hidroxilase/antagonistas & inibidores , Temperatura , Relação Dose-Resposta a Droga , Células Hep G2 , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Indóis/química , Estrutura Molecular , Panobinostat , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/metabolismo , Relação Estrutura-AtividadeRESUMO
We extended thermal proteome profiling to detect transmembrane protein-small molecule interactions in cultured human cells. When we assessed the effects of detergents on ATP-binding profiles, we observed shifts in denaturation temperature for ATP-binding transmembrane proteins. We also observed cellular thermal shifts in pervanadate-induced T cell-receptor signaling, delineating the membrane target CD45 and components of the downstream pathway, and with drugs affecting the transmembrane transporters ATP1A1 and MDR1.
Assuntos
Proteínas de Membrana/metabolismo , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Células CACO-2 , Temperatura Alta , Humanos , Células Jurkat , Células K562 , Ligantes , Ligação Proteica , Estabilidade Proteica , Proteoma/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Vanadatos/farmacologiaRESUMO
The direct detection of drug-protein interactions in living cells is a major challenge in drug discovery research. Recently, we introduced an approach termed thermal proteome profiling (TPP), which enables the monitoring of changes in protein thermal stability across the proteome using quantitative mass spectrometry. We determined the intracellular thermal profiles for up to 7,000 proteins, and by comparing profiles derived from cultured mammalian cells in the presence or absence of a drug we showed that it was possible to identify direct and indirect targets of drugs in living cells in an unbiased manner. Here we demonstrate the complete workflow using the histone deacetylase inhibitor panobinostat. The key to this approach is the use of isobaric tandem mass tag 10-plex (TMT10) reagents to label digested protein samples corresponding to each temperature point in the melting curve so that the samples can be analyzed by multiplexed quantitative mass spectrometry. Important steps in the bioinformatic analysis include data normalization, melting curve fitting and statistical significance determination of compound concentration-dependent changes in protein stability. All analysis tools are made freely available as R and Python packages. The workflow can be completed in 2 weeks.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Espectrometria de Massas , Proteoma/genética , Humanos , Células K562 , Análise Serial de Proteínas , Estabilidade Proteica , TemperaturaRESUMO
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 E. coli tryptic digests.
Assuntos
Proteínas de Escherichia coli/química , Fragmentos de Peptídeos/isolamento & purificação , Proteômica/normas , Coloração e Rotulagem/normas , Espectrometria de Massas em Tandem/normas , Algoritmos , Escherichia coli/química , Humanos , Células Jurkat , Células K562 , Peso Molecular , Fragmentos de Peptídeos/química , Proteômica/métodos , Coloração e Rotulagem/métodosRESUMO
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 ((15)N, (13)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 (15)N- and (13)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, (12)C(8)H(16)(15)N(1)(+); TMT127H, (12)C(7)(13)C(1)H(16)(14)N(1)(+); TMT129L, (12)C(6)(13)C(2)H(16)(15)N(1)(+); and TMT129H, (12)C(5)(13)C(3)H(16)(14)N(1)(+)). 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.
Assuntos
Espectrometria de Massas em Tandem/métodos , Dasatinibe , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Indicadores e Reagentes/química , Células K562 , Gravidez , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologiaRESUMO
We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.