Abstract
Robust isolation and identification of peptides phosphorylated at their tyrosine residues are key steps in deciphering complex signaling networks governed by protein tyrosine kinases, including kinases involved in oncogenesis. Phosphotyrosine (pY)-containing peptides are commonly isolated from cellular lysates by means of antibody and/or metal affinity-based enrichment followed by their identification by mass spectrometry. Herein, we describe robust two-stage isolation of phosphotyrosine peptides and mass spectrometry-aided identification of phosphosites to characterize basal signaling networks in unstimulated non-small cell lung cancer (NSCLC) cell lines.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Yoshida T, Zhang G, Smith MA et al (2014) Tyrosine phosphoproteomics identified both co-drivers and co-targeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer. Clin Cancer Res 20:4059–4074
Huang PH, Mukasa A, Bonavia R et al (2007) Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proc Natl Acad Sci U S A 104:12867–12872
Iwai LK, Payne LS, Luczynski MT et al (2013) Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants. Biochem J 454:501–513
Hunter T (2009) Tyrosine phosphorylation: thirty years and counting. Curr Opin Cell Biol 21:140–146
Bergström LS, Molin M, Savitski MM et al (2008) Immunoaffinity enrichments followed by mass spectrometric detection for studying global protein tyrosine phosphorylation. J Proteome Res 7:2897–2910
Lind SB, Artemenko KA, Pettersson U (2012) A strategy for identification of protein tyrosine phosphorylation. Methods 56:275–283
Lombardi B, Rendell N, Edwards M, Katan M, Zimmermann JG (2015) Evaluation of phosphopeptide enrichment strategies for quantitative TMT analysis of complex network dynamics in cancer-associated cell signalling. EuPA Open Proteom 1:10–15
Zhang Y, Wolf-Yadlin A, Ross PL et al (2005) Time-resolved phosphotyrosine analysis reveals dynamic modules in EGFR signaling. Mol Cell Proteomics 4:1240–1250
Boersema PJ, Mohammed S, Heck AJ (2009) Phosphopeptide fragmentation and analysis by mass spectrometry. J Mass Spectrom 44:861–878
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372
Ficarro SB, Adelmant G, Tomar MN et al (2009) Magnetic bead processor for rapid evaluation and optimization of parameters for phosphopeptide enrichment. Anal Chem 81:4566–4575
Acknowledgments
This work was supported by the Institute of Cancer Research and Cancer Research UK [C36478/A19281].
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
Broncel, M., Huang, P.H. (2017). Analysis of Phosphotyrosine Signaling Networks in Lung Cancer Cell Lines. In: Tan, AC., Huang, P. (eds) Kinase Signaling Networks. Methods in Molecular Biology, vol 1636. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7154-1_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7154-1_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7152-7
Online ISBN: 978-1-4939-7154-1
eBook Packages: Springer Protocols