Phosphotyrosine-based-phosphoproteomics scaled-down to biopsy level for analysis of individual tumor biology and treatment selection

Labots, Mariëtte; Mijn, Johannes C. van der; Beekhof, Robin; Piersma, Sander R.; Haas, Richard R. de; Pham, Thang V.; Knol, Jaco C.; Dekker, Henk; Grieken, Nicole C.T. van; Verheul, Henk M.W.; Jiménez, Connie R.

doi:10.1016/j.jprot.2017.04.014

Cited by 31 publications

(29 citation statements)

References 38 publications

(56 reference statements)

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“…A next stage is to apply INKA to more advanced cancer models and, especially, clinical samples. To analyze limited amounts of patient tumor tissue in a clinical practice setting, we have recently downscaled tyrosine phosphoproteomics to clinical needle‐biopsy levels (Labots et al , ). Using this workflow, we showed the feasibility of phosphoproteomics combined with INKA analysis of patient samples from a clinical molecular profiling study with kinase inhibitors (Labots et al , in preparation), showing a reduced INKA score of EGFR upon erlotinib treatment.…”

Section: Discussionmentioning

confidence: 99%

“…For tumor biopsy phosphoproteomics in this study, needle biopsies from a patient with advanced head and neck squamous cell carcinoma, obtained in an Institutional Review Board‐approved molecular profiling study before and after 2 weeks of treatment with erlotinib (NCT clinical trials identifier 01636908; http://www.clinicaltrials.gov), were processed as described elsewhere (Labots et al , ). For phosphoproteomics, a 2.5 mg protein equivalent was used.…”

Section: Methodsmentioning

confidence: 99%

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INKA , an integrative data analysis pipeline for phosphoproteomic inference of active kinases

Beekhof

Alphen

Henneman

et al. 2019

Molecular Systems Biology

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Identifying hyperactive kinases in cancer is crucial for individualized treatment with specific inhibitors. Kinase activity can be discerned from global protein phosphorylation profiles obtained with mass spectrometry‐based phosphoproteomics. A major challenge is to relate such profiles to specific hyperactive kinases fueling growth/progression of individual tumors. Hitherto, the focus has been on phosphorylation of either kinases or their substrates. Here, we combined label‐free kinase‐centric and substrate‐centric information in an Integrative Inferred Kinase Activity ( INKA ) analysis. This multipronged, stringent analysis enables ranking of kinase activity and visualization of kinase–substrate networks in a single biological sample. To demonstrate utility, we analyzed (i) cancer cell lines with known oncogenes, (ii) cell lines in a differential setting (wild‐type versus mutant, +/− drug), (iii) pre‐ and on‐treatment tumor needle biopsies, (iv) cancer cell panel with available drug sensitivity data, and (v) patient‐derived tumor xenografts with INKA ‐guided drug selection and testing. These analyses show superior performance of INKA over its components and substrate‐based single‐sample tool KARP , and underscore target potential of high‐ranking kinases, encouraging further exploration of INKA 's functional and clinical value.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

INKA , an integrative data analysis pipeline for phosphoproteomic inference of active kinases

Beekhof

Alphen

Henneman

et al. 2019

Molecular Systems Biology

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“…The potential of this high-throughput method has first been evidenced by the identification of the Anaplastic lymphoma kinase (ALK), Reactive oxygen species (ROS) and Platelet derived growth factor alpha (PDGFRα) mediated Non-small cell lung cancer (NSCLC) subtypes in 2007 [11] and just recently by identification of 6 kinases prognostic for the outcome of triple negative breast cancer [12]. We previously demonstrated that MS-based profiling of the tyrosine phosphoproteome in tumor biopsies is feasible and provides patient-specific profiles, enabling its further development for treatment selection purposes [13]. In the preclinical setting, for example using chemical proteomics in triple negative breast cancer cell lines [14], alterations of the kinome in response to PKI inhibition have been shown.…”

Section: Introductionmentioning

confidence: 99%

Kinase Inhibitor Treatment of Patients with Advanced Cancer Results in High Tumor Drug Concentrations and in Specific Alterations of the Tumor Phosphoproteome

Labots

Pham

Honeywell

et al. 2020

Cancers

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Identification of predictive biomarkers for targeted therapies requires information on drug exposure at the target site as well as its effect on the signaling context of a tumor. To obtain more insight in the clinical mechanism of action of protein kinase inhibitors (PKIs), we studied tumor drug concentrations of protein kinase inhibitors (PKIs) and their effect on the tyrosine-(pTyr)-phosphoproteome in patients with advanced cancer. Tumor biopsies were obtained from 31 patients with advanced cancer before and after 2 weeks of treatment with sorafenib (SOR), erlotinib (ERL), dasatinib (DAS), vemurafenib (VEM), sunitinib (SUN) or everolimus (EVE). Tumor concentrations were determined by LC-MS/MS. pTyr-phosphoproteomics was performed by pTyr-immunoprecipitation followed by LC-MS/MS. Median tumor concentrations were 2–10 µM for SOR, ERL, DAS, SUN, EVE and >1 mM for VEM. These were 2–178 × higher than median plasma concentrations. Unsupervised hierarchical clustering of pTyr-phosphopeptide intensities revealed patient-specific clustering of pre- and on-treatment profiles. Drug-specific alterations of peptide phosphorylation was demonstrated by marginal overlap of robustly up- and downregulated phosphopeptides. These findings demonstrate that tumor drug concentrations are higher than anticipated and result in drug specific alterations of the phosphoproteome. Further development of phosphoproteomics-based personalized medicine is warranted.

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“…β ‐casein and BSA/ β ‐casein mixture) and also human serum. Phosphoproteome analysis in biological fluids has attracted significant attention in recent years since a linkage between the phosphorylated forms of proteins/peptides and tumor formation has been shown for different tumor types (Gökmen et al, ; Hong et al, ; Labots et al, ; Lin et al, ; Zhao et al, ; Zhao et al, ). Moreover, in our case, the use of a capillary monolith with larger diameter and shorter length allowed us to work with relatively lower back pressures (i.e.…”

Section: Resultsmentioning

confidence: 99%

Ti (IV) attached‐phosphonic acid functionalized capillary monolith as a stationary phase for in‐syringe‐type fast and robust enrichment of phosphopeptides

Salimi

Kip

Çelikbıçak

et al. 2019

Biomedical Chromatography

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In this study, poly(vinylphosphonic acid-co-ethylene dimethacrylate), poly(VPA-co-EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 μm. The phosphopeptides from different sources were enriched on Ti (IV)-attached poly(VPA-co-EDMA) monolith using a syringe-pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of β-casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA-co-EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of β-casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal-to-noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA-co-EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.KEYWORDS capillary monolith, human serum, immobilized metal affinity chromatography, phosphopeptide enrichment, syringe type microextration, vinylphosphonic acid

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Phosphotyrosine-based-phosphoproteomics scaled-down to biopsy level for analysis of individual tumor biology and treatment selection

Cited by 31 publications

References 38 publications

INKA , an integrative data analysis pipeline for phosphoproteomic inference of active kinases

INKA , an integrative data analysis pipeline for phosphoproteomic inference of active kinases

Kinase Inhibitor Treatment of Patients with Advanced Cancer Results in High Tumor Drug Concentrations and in Specific Alterations of the Tumor Phosphoproteome

Ti (IV) attached‐phosphonic acid functionalized capillary monolith as a stationary phase for in‐syringe‐type fast and robust enrichment of phosphopeptides

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