Abstract
Dynamic tyrosine phosphorylation is a key molecular modulation for many signal transduction events. Because of their low abundance and dynamic nature in cells, the detection and enrichment of phosphotyrosine proteins has long relied on specific antibodies, such as 4G10 and P-Tyr-100. Another well-established approach for phosphotyrosine proteins recognition and enrichment is by their specific binding domains, such as Src homology 2 (SH2) domains. In this chapter, we describe a typical analytical approach for purifying specific SH2 domains, enriching specific phosphotyrosine proteins from activated cells, mass spectrometry analysis, and related data analysis.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Sadowski I, Stone JC, Pawson T (1986) A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps. Mol Cell Biol 6:4396–4408
Seet BT, Dikic I, Zhou MM, Pawson T (2006) Reading protein modifications with interaction domains. Nat Rev Mol Cell Biol 7:473–483
Bensimon A, Heck AJ, Aebersold R (2012) Mass spectrometry-based proteomics and network biology. Annu Rev Biochem 81:379–405
Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H et al (2007) Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131:1190–1203
Wolf-Yadlin A, Hautaniemi S, Lauffenburger DA, White FM (2007) Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks. Proc Natl Acad Sci U S A 104:5860–5865
Jorgensen C, Sherman A, Chen GI, Pasculescu A, Poliakov A, Hsiung M et al (2009) Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells. Science 326:1502–1509
Pawson T, Gish GD, Nash P (2001) SH2 domains, interaction modules and cellular wiring. Trends Cell Biol 11:504–511
Liu BA, Jablonowski K, Raina M, Arce M, Pawson T, Nash PD (2006) The human and mouse complement of SH2 domain proteins-establishing the boundaries of phosphotyrosine signaling. Mol Cell 22:851–868
Liu BA, Shah E, Jablonowski K, Stergachis A, Engelmann B, Nash PD (2011) The SH2 domain-containing proteins in 21 species establish the provenance and scope of phosphotyrosine signaling in eukaryotes. Sci Signal 4:ra83
Machida K, Thompson CM, Dierck K, Jablonowski K, Karkkainen S, Liu B et al (2007) High-throughput phosphotyrosine profiling using SH2 domains. Mol Cell 26:899–915
Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG et al (1993) SH2 domains recognize specific phosphopeptide sequences. Cell 72:767–778
Tinti M, Kiemer L, Costa S, Miller ML, Sacco F, Olsen JV et al (2013) The SH2 domain interaction landscape. Cell Rep 3:1293–1305
Engelmann BW, Kim Y, Wang M, Peters B, Rock RS, Nash PD (2014) The development and application of a quantitative peptide microarray based approach to protein interaction domain specificity space. Mol Cell Proteomics 13:3647–3662
Kaushansky A, Allen JE, Gordus A, Stiffler MA, Karp ES, Chang BH et al (2010) Quantifying protein-protein interactions in high throughput using protein domain microarrays. Nat Protoc 5:773–790
Liu H, Li L, Voss C, Wang F, Liu J, Li SS (2015) A comprehensive immunoreceptor phosphotyrosine-based signaling network revealed by reciprocal protein-peptide array screening. Mol Cell Proteomics 14:1846–1858
Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M (2003) A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol 21:315–318
Uezu A, Okada H, Murakoshi H, del Vescovo CD, Yasuda R, Diviani D et al (2012) Modified SH2 domain to phototrap and identify phosphotyrosine proteins from subcellular sites within cells. Proc Natl Acad Sci U S A 109:E2929–E2938
Bisson N, James DA, Ivosev G, Tate SA, Bonner R, Taylor L et al (2011) Selected reaction monitoring mass spectrometry reveals the dynamics of signaling through the GRB2 adaptor. Nat Biotechnol 29:653–658
Acknowledgment
This work was supported by South University of Science and Technology of China basic research fund to R.T. (FRG-SUSTC1501A-19) and National Natural Science Foundation of China (21575057).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Ke, M., Chu, B., Lin, L., Tian, R. (2017). SH2 Domains as Affinity Reagents for Phosphotyrosine Protein Enrichment and Proteomic Analysis. In: Machida, K., Liu, B. (eds) SH2 Domains. Methods in Molecular Biology, vol 1555. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6762-9_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6762-9_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6760-5
Online ISBN: 978-1-4939-6762-9
eBook Packages: Springer Protocols