Abstract
A precisely controlled network of protein–protein interactions constitutes the basis for functional signaling pathways. This equilibrium is more often than not disrupted in cancer cells, by the aberrant expression or activation of oncogenic proteins. Therefore, the analysis of protein interaction networks in cancer cells has become crucial to expand our comprehension of the molecular underpinnings of tumor formation and progression. This protocol describes a sample preparation method for the analysis of signaling complexes by mass spectrometry (MS), following the affinity purification of a protein of interest from a cancer cell line or a solid tumor. In particular, we provide a spin tip-based protease digestion procedure that offers a more rapid and controlled alternative to other gel-based and gel-free methods. This sample preparation protocol represents a useful strategy to identify protein interactions and to gain insight into the molecular mechanisms that contribute to a given cancer phenotype.
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References
Gibson TJ (2009) Cell regulation: determined to signal discrete cooperation. Trends Biochem Sci 34:471–482
Pawson T (1995) Protein modules and signalling networks. Nature 373:573–580
Goh KI, Cusick ME, Valle D, Childs B, Vidal M, Barabasi AL (2007) The human disease network. Proc Natl Acad Sci U S A 104:8685–8690
Menche J, Sharma A, Kitsak M, Ghiassian SD, Vidal M, Loscalzo J, Barabasi AL (2015) Disease networks Uncovering disease-disease relationships through the incomplete interactome. Science 347:1257601
Lambert JP, Ivosev G, Couzens AL, Larsen B, Taipale M, Lin ZY et al (2013) Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition. Nat Methods 10:1239–1245
Dunham WH, Mullin M, Gingras AC (2012) Affinity-purification coupled to mass spectrometry: basic principles and strategies. Proteomics 12:1576–1590
Gamblin CL, Hardy EJ, Chartier FJ, Bisson N, Laprise P (2014) A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity. J Cell Biol 204:487–495
Bisson N, James DA, Ivosev G, Tate SA, Bonner R, Taylor L, Pawson T (2011) Selected reaction monitoring mass spectrometry reveals the dynamics of signaling through the GRB2 adaptor. Nat Biotechnol 29:653–658
So J, Pasculescu A, Dai AY, Williton K, James A, Nguyen V et al (2015) Integrative analysis of kinase networks in TRAIL-induced apoptosis provides a source of potential targets for combination therapy. Sci Signal 8:rs3
Filteau M, Diss G, Torres-Quiroz F, Dube AK, Schraffl A, Bachmann VA et al (2015) Systematic identification of signal integration by protein kinase A. Proc Natl Acad Sci U S A 112:4501–4506
Choi H, Larsen B, Lin ZY, Breitkreutz A, Mellacheruvu D, Fermin D et al (2011) SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat Methods 8:70–73
Acknowledgements
The spin tip digestion method was initially developed in the laboratory of Dr. Tony Pawson at the Lunenfeld-Tanenbaum Research Institute, with input from Karen Colwill, Brett Larsen and Vivian Nguyen. This work was supported by grants from the Canadian Institutes for Health Research (MOP-130335) and the Canada Foundation for Innovation (32308) (to N.B.). A.B. and L.V. held Pierre J. Durand Scholarships from Université Laval Faculty of Medicine, and L.V. currently holds the Louis-Poirier Scholarship and a Université Laval Cancer Research Centre scholarship. N.B. was a scholar of the “Fonds de Recherche du Québec-Santé” in partnership with the Quebec Breast Cancer Foundation and holds a Canada Research Chair in Cancer Proteomics.
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Beigbeder, A., Vélot, L., James, D.A., Bisson, N. (2016). Sample Preparation for Mass Spectrometry Analysis of Protein–Protein Interactions in Cancer Cell Lines and Tissues. In: Ursini-Siegel, J., Beauchemin, N. (eds) The Tumor Microenvironment. Methods in Molecular Biology, vol 1458. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3801-8_23
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DOI: https://doi.org/10.1007/978-1-4939-3801-8_23
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