23 research outputs found
Probing SH2-domains using Inhibitor Affinity Purification (IAP)
Höfener M, Heinzlmeir S, Kuster B, Sewald N. Probing SH2-domains using Inhibitor Affinity Purification (IAP). Proteome Science. 2014;12(1): 41.Background
Many human diseases are correlated with the dysregulation of signal transduction processes. One of the most important protein interaction domains in the context of signal transduction is the Src homology 2 (SH2) domain that binds phosphotyrosine residues. Hence, appropriate methods for the investigation of SH2 proteins are indispensable in diagnostics and medicinal chemistry. Therefore, an affinity resin for the enrichment of all SH2 proteins in one experiment would be desirable. However, current methods are unable to address all SH2 proteins simultaneously with a single compound or a small array of compounds.
Results
In order to overcome these limitations for the investigation of this particular protein family in future experiments, a dipeptide-derived probe has been designed, synthesized and evaluated. This probe successfully enriched 22 SH2 proteins from mixed cell lysates which contained 50 SH2 proteins. Further characterization of the SH2 binding properties of the probe using depletion and competition experiments indicated its ability to enrich complexes consisting of SH2 domain bearing regulatory PI3K subunits and catalytic phosphoinositide 3-kinase (PI3K) subunits that have no SH2 domain.
Conclusion
The results make this probe a promising starting point for the development of a mixed affinity resin with complete SH2 protein coverage. Moreover, the additional findings render it a valuable tool for the evaluation of PI3K complex interrupting inhibitors
Pharmacoproteomic characterisation of human colon and rectal cancer
Most molecular cancer therapies act on protein targets but data on the proteome status of patients and cellular models for proteome-guided pre-clinical drug sensitivity studies are only beginning to emerge. Here, we profiled the proteomes of 65 colorectal cancer (CRC) cell lines to a depth of > 10,000 proteins using mass spectrometry. Integration with proteomes of 90 CRC patients and matched transcriptomics data defined integrated CRC subtypes, highlighting cell lines representative of each tumour subtype. Modelling the responses of 52 CRC cell lines to 577 drugs as a function of proteome profiles enabled predicting drug sensitivity for cell lines and patients. Among many novel associations, MERTK was identified as a predictive marker for resistance towards MEK1/2 inhibitors and immunohistochemistry of 1,074 CRC tumours confirmed MERTK as a prognostic survival marker. We provide the proteomic and pharmacological data as a resource to the community to, for example, facilitate the design of innovative prospective clinical trials. © 2017 The Authors. Published under the terms of the CC BY 4.0 licens
Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC-MS/MS
Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC-MS/MS using a 1x150 mm column shows excellent reproducibility of chromatographic retention time (2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC-MS/MS is suitable for a broad range of proteomic applications
The formation of a camalexin-biosynthetic metabolon
Arabidopsis thaliana efficiently synthesizes the antifungal phytoalexin camalexin without apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting channeling of the biosynthetic pathway by formation of an enzyme complex. To identify such protein interactions, two independent untargeted co49 immunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B1 and CYP71A13 as baits were performed and the camalexin biosynthetic P450 enzymes were shown to co-purify. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, interaction of CYP71A13 and Arabidopsis P450 Reductase 1 (ATR1) was observed. An increased substrate affinity of CYP79B2 in presence of CYP71A13 was shown, indicating allosteric interaction. Camalexin biosynthesis involves glutathionylation of an intermediary indole-3-cyanohydrin, synthesized by CYP71A12 and especially CYP71A13. It was demonstrated by FRET-FLIM and co-IP, that the glutathione transferase GSTU4, which is co-expressed with tryptophan- and camalexin-specific enzymes, was physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knock-out and reduced in GSTU4 overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather has a role in a competing mechanism
New Affinity Probe Targeting VEGF Receptors for Kinase Inhibitor Selectivity Profiling by Chemical Proteomics
Solid
tumors are dependent for growth on nutrients and the supply
of oxygen, which they often acquire via neoangiogenesis. Vascular
endothelial growth factors and the corresponding receptors (VEGFRs)
play central roles in this process, and consequently, the blockade
of this pathway is one therapeutic strategy for cancer treatment.
A number of small molecules inhibiting VEGFR inhibitors have been
developed for clinical use, and a comprehensive view of target selectivity
is important to assess the therapeutic as well as risk potential of
a drug molecule. Recent advances in mass spectrometry-based chemical
proteomics allow analyses of drug–target interactions under
close-to-physiological conditions, and in this study, we report on
the design, synthesis, and application of a small molecule affinity
probe as a tool for the selectivity profiling of VEGFR and other kinase
inhibitors. The probe is capable of binding >132 protein kinases,
including angiokinases such as VEGFRs, PDGFRs, and c-KIT from lysates
of cancer cell lines or human placenta tissue. Combining the new probe
with Kinobeads in competitive binding assays, we were able to identify
nanomolar off-targets of the VEGFR/PDGFR inhibitors pazopanib and
axitinib. Because of its broad binding spectrum, the developed chemical
tool can be generically used for the discovery of kinase inhibitor
targets, which may contribute to a more comprehensive understanding
of the mechanisms of action of such drugs
Evaluation of Kinase Activity Profiling Using Chemical Proteomics
Protein
kinases are important mediators of intracellular signaling
and are reversibly activated by phosphorylation. Immobilized kinase
inhibitors can be used to enrich these often low-abundance proteins,
to identify targets of kinase inhibitors, or to probe their selectivity.
It has been suggested that the binding of kinases to affinity beads
reflects a kinase’s activation status, a concept that is under
considerable debate. To assess the merits of the idea, we performed
a series of experiments including quantitative phosphoproteomics and
purification of kinases by single or mixed affinity matrices from
signaling activated or resting cancer cells. The data show that mixed
affinity beads largely bind kinases independent of their activation
status, and experiments using individual immobilized kinase inhibitors
show mixed results in terms of preference for binding the active or
inactive conformation. Taken together, activity- or conformation-dependent
binding to such affinity resins depends (i) on the kinase, (ii) on
the affinity probe, and (iii) on the activation status of the lysate
or cell. As a result, great caution should be exercised when inferring
kinase activity from such binding data. The results also suggest that
assaying kinase activity using binding data is restricted to a limited
number of well-chosen cases
Evaluation of Kinase Activity Profiling Using Chemical Proteomics
Protein
kinases are important mediators of intracellular signaling
and are reversibly activated by phosphorylation. Immobilized kinase
inhibitors can be used to enrich these often low-abundance proteins,
to identify targets of kinase inhibitors, or to probe their selectivity.
It has been suggested that the binding of kinases to affinity beads
reflects a kinase’s activation status, a concept that is under
considerable debate. To assess the merits of the idea, we performed
a series of experiments including quantitative phosphoproteomics and
purification of kinases by single or mixed affinity matrices from
signaling activated or resting cancer cells. The data show that mixed
affinity beads largely bind kinases independent of their activation
status, and experiments using individual immobilized kinase inhibitors
show mixed results in terms of preference for binding the active or
inactive conformation. Taken together, activity- or conformation-dependent
binding to such affinity resins depends (i) on the kinase, (ii) on
the affinity probe, and (iii) on the activation status of the lysate
or cell. As a result, great caution should be exercised when inferring
kinase activity from such binding data. The results also suggest that
assaying kinase activity using binding data is restricted to a limited
number of well-chosen cases