Abstract
Phosphoproteomics is an important tool for the unbiased investigation of signaling network activation and has particular application to unraveling aberrant signaling driving cancer progression. However, validating the behavior of specific phosphosites across multiple experimental conditions remains challenging, due to limitations inherent in discovery-based proteomic workflows and the limited availability of high-quality antibodies required for alternative, immunoaffinity-based methods. Targeted phosphoproteomics enables specific phosphosites to be quantified reproducibly across multiple experimental conditions. Importantly, targeted phosphoproteomic assays can be designed rapidly on the basis of data acquired in discovery proteomic experiments and circumvent the requirement of immunoaffinity techniques for reliable antibodies raised to specific, potentially poorly immunogenic phosphopeptides. In the following protocol, we present a method for the relative quantification of phosphosites across multiple experimental conditions and/or technical and biological replicates.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Tabb DL, Lorenzo Vega-Montoto L, Rudnick PA, Variyath AM, Ham AJ, Bunk DM, Kilpatrick LE, Billheimer DD, Blackman RK, Cardasis HL, Carr SA, Clauser KR, Jaffe JD, Kowalski KA, Neubert TA, Regnier FE, Schilling B, Tegeler TJ, Wang M, Wang P, Whiteaker JR, Zimmerman LJ, Fisher SJ, Gibson BW, Kinsinger CR, Mesri M, Rodriguez H, Stein SE, Tempst P, Paulovich AG, Liebler DC, Spiegelman C (2010) Repeatability and reproducibility in proteomic identifications by liquid chromatography–tandem mass spectrometry. J Proteome Res 9:761–776
Baker M (2015) Reproducibility crisis: blame it on the antibodies. Nature 521:274–276
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
Curran TG, Zhang Y, Ma DJ, Sarkaria JN, White FM (2015) MARQUIS: a multiplex method for absolute quantification of peptides and post-translational modifications. Nat Commun 6:5924. doi:10.1038/ncomms692
Iwai LK, Payne LS, Luczynski MT, Chang F, Xu H, Clinton RW, Paul A, Esposito EA, Gridley S, Leitinger B, Naegle KM, Huang PH (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
Corallino S, Iwai LK, Payne LS, Huang PH, Sacco F, Cesareni G, Castagnoli L (2015) Alterations in the phosphoproteomic profile of cells expressing a non-functional form of the SHP2 phosphatase. New Biotechnol 33(5 Pt A):524–536. doi:10.1016/j.nbt.2015.08.002
Mead JA, Luca Bianco L, Ottone V, Barton C, Richard G, Kay RG, Lilley KS, Bond NJ, Bessant C (2009) MRMaid, the web-based tool for designing multiple reaction monitoring (MRM) transitions. Mol Cell Proteomics 8:696–705
Brendan MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, Kern R, Tabb DL, Liebler DC, MacCoss MJ (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26:966–968
Colangelo MC, Chung L, Can Bruce C, Cheung K (2013) Review of software tools for design and analysis of large scale MRM proteomic datasets. Methods 61:287–298
Acknowledgment
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
Payne, L.S., Huang, P.H. (2017). Targeted Analysis of Phosphotyrosine Signaling by Multiple Reaction Monitoring Mass Spectrometry. 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_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7154-1_17
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