title: Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry.
abstract: 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.
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Franken
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Toby
authors.initials: T
authors.lastname: Mathieson
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.|Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Dorothee
authors.initials: D
authors.lastname: Childs
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Gavain M A
authors.initials: GM
authors.lastname: Sweetman
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Werner
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Tögel
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Carola
authors.initials: C
authors.lastname: Doce
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Gade
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Marcus
authors.identifier: http://orcid.org/0000-0002-8343-8977
authors.initials: M
authors.lastname: Bantscheff
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Gerard
authors.initials: G
authors.lastname: Drewes
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Friedrich B M
authors.initials: FB
authors.lastname: Reinhard
authors.affiliation: Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Wolfgang
authors.identifier: http://orcid.org/0000-0002-0474-2218
authors.initials: W
authors.lastname: Huber
authors.affiliation: Cellzome, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.
authors.forename: Mikhail M
authors.initials: MM
authors.lastname: Savitski
mesh_terms: D016503:Drug Delivery Systems / Q000379:methods*; D006801:Humans; D020014:K562 Cells; D013058:Mass Spectrometry; D040081:Protein Array Analysis; D055550:Protein Stability; D020543:Proteome / Q000235:genetics*; D013696:Temperature
chemical_list: D020543:Proteome