Evaluation of phosphopeptide enrichment strategies for quantitative TMT analysis of complex network dynamics in cancer-associated cell signalling

Lombardi, Benedetta; Rendell, Nigel B.; Edwards, Meredith; Katan, Matilda; Zimmermann, Jasminka Godovac

doi:10.1016/j.euprot.2015.01.002

Cited by 15 publications

(19 citation statements)

References 27 publications

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“…In our phosphoproteomic study, we identified nine distinct Kif26b phosphorylation sites on eight unique phosphopeptides, and all these sites mapped to serine or threonine residues ( Figure 1—figure supplement 2 and Supplementary file 1 ). In general, tyrosine phosphorylation is more dynamic than serine or threonine phosphorylation and tends to be underrepresented in large-scale phosphoproteomic studies ( Lombardi et al, 2015 ). We therefore examined whether Wnt5a-Ror signaling induces tyrosine phosphorylation of Kif26b in NIH/3T3 cells using an anti-phosphotyrosine antibody-based affinity pull-down approach.…”

Section: Discussionmentioning

confidence: 99%

Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates

Susman

Karuna

Kunz

et al. 2017

eLife

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Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/β-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.

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Section: Discussionmentioning

confidence: 99%

Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates

Susman

Karuna

Kunz

et al. 2017

eLife

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“…Amongst the methodologies available to enrich phosphopeptides in MM, the most widely practiced are immobilised metal ion chromatography (IMAC) (Thingholm and Larsen 2016a) and titanium dioxide (Thingholm and Larsen 2016b), and combinations thereof. In addition, fractionation protocols such as strong cation exchange (SCX) (Lombardi et al 2015), Fig. 10 Illustration of pathway signalling where phosphorylation signalling PTMs have been sequenced and annotated in melanoma tumours from patients hydrophilic interaction liquid chromatography (HILIC) (Boersema et al 2008) or basic reversed-phase chromatography can be utilised to increase coverage of the phosphoproteome (Batth et al 2014).…”

Section: Pathway Signalling and Protein Phosphorylationmentioning

confidence: 99%

Clinical protein science in translational medicine targeting malignant melanoma

et al. 2019

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Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular

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“…Enrichment of the fraction of phosphorylated peptides utilised antibody precipitation coupled with TiO2 chromatography as described previously [ 26 ], with minor changes. For the anti-phosphotytrosine peptide immunoprecipitation, the antibody PT66 was replaced with pY1000 (Cell Signalling Technology, Hitchin, Hertfordshire, UK).…”

Section: Methodsmentioning

confidence: 99%

“…LC-MS/MS analysis was performed using an LTQ-Velos Orbitrap mass spectrometer (Thermo Fisher Scientific) as described previously [ 26 , 47 ]. A detailed description of data analysis can be found in the Supplementary Materials and Methods .…”

Section: Methodsmentioning

confidence: 99%

“…We use quantitative mass spectrometry (MS)-based phosphoproteomics methods that have emerged in recent years as a powerful approach to study phosphorylation dynamics in cell cultures and tissues [ 25 ]. As previously described, we adapted these methods to allow analysis of small amounts of TERT-NHUC cells [ 26 ]. To further develop existing approaches for data analysis [ 27 – 29 ], we assemble pathway and network bioinformatics tools in a novel protocol that reveals new signalling links unique to the FGFR3-TACC3 fusion.…”

Section: Introductionmentioning

confidence: 99%

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Unique signalling connectivity of FGFR3-TACC3 oncoprotein revealed by quantitative phosphoproteomics and differential network analysis

et al. 2017

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The FGFR3-TACC3 fusion is an oncogenic driver in diverse malignancies, including bladder cancer, characterized by upregulated tyrosine kinase activity. To gain insights into distinct properties of FGFR3-TACC3 down-stream signalling, we utilised telomerase-immortalised normal human urothelial cell lines expressing either the fusion or wild-type FGFR3 (isoform IIIb) for subsequent quantitative proteomics and network analysis. Cellular lysates were chemically labelled with isobaric tandem mass tag reagents and, after phosphopeptide enrichment, liquid chromatography-high mass accuracy tandem mass spectrometry (LC-MS/MS) was used for peptide identification and quantification. Comparison of data from the two cell lines under non-stimulated and FGF1 stimulated conditions and of data representing physiological stimulation of FGFR3 identified about 200 regulated phosphosites. The identified phosphoproteins and quantified phosphosites were further analysed in the context of functional biological networks by inferring kinase-substrate interactions, mapping these to a comprehensive human signalling interaction network, filtering based on tissue-expression profiles and applying disease module detection and pathway enrichment methods. Analysis of our phosphoproteomics data using these bioinformatics methods combined into a new protocol—Disease Relevant Analysis of Genes On Networks (DRAGON)—allowed us to tease apart pathways differentially involved in FGFR3-TACC3 signalling in comparison to wild-type FGFR3 and to investigate their local phospho-signalling context. We highlight 9 pathways significantly regulated only in the cell line expressing FGFR3-TACC3 fusion and 5 pathways regulated only by stimulation of the wild-type FGFR3. Pathways differentially linked to FGFR3-TACC3 fusion include those related to chaperone activation and stress response and to regulation of TP53 expression and degradation that could contribute to development and maintenance of the cancer phenotype.

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Evaluation of phosphopeptide enrichment strategies for quantitative TMT analysis of complex network dynamics in cancer-associated cell signalling

Cited by 15 publications

References 27 publications

Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates

Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates

Clinical protein science in translational medicine targeting malignant melanoma

Unique signalling connectivity of FGFR3-TACC3 oncoprotein revealed by quantitative phosphoproteomics and differential network analysis

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