Abstract
A number of challenges have to be overcome to identify a complete complement of phosphorylated proteins, the phosphoproteome, from cells and tissues. Phosphorylated proteins are typically of low abundance and moreover, the proportion of phosphorylated sites on a given protein is generally low. The challenge is further compounded when the tissue from which protein can be recovered is limited. Global phosphoproteomics primarily relies on efficient enrichment methods for phosphopeptides involving affinity binding coupled with analysis by fast high-resolution mass spectrometry (MS) and subsequent identification using various software packages. Here, we describe an effective protocol for phosphopeptide enrichment using an Iron-IMAC resin in combination with titanium dioxide (TiO2) beads from trypsin digested protein samples of the filamentous fungus Magnaporthe oryzae. Representative protocols for LC-MS/MS analysis and phosphopeptide identification are also described.
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
Khoury GA, Baliban RC, Floudas CA (2011) Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Sci Rep 1:90. https://doi.org/10.1038/srep00090
Yachie N, Saito R, Sugiyama N et al (2011) Integrative features of the yeast phosphoproteome and protein-protein interaction map. PLoS Comput Biol 7:e1001064. https://doi.org/10.1371/journal.pcbi.1001064
Wilhelm M, Schlegl J, Hahne H et al (2014) Mass-spectrometry-based draft of the human proteome. Nature 509:582–587. https://doi.org/10.1038/nature13319
Zhou H, Di Palma S, Preisinger C et al (2013) Toward a comprehensive characterization of a human Cancer cell Phosphoproteome. J Proteome Res 12:260–271. https://doi.org/10.1021/pr300630k
Rosenberg A, Soufi B, Ravikumar V et al (2015) Phosphoproteome dynamics mediate revival of bacterial spores. BMC Biol 13:76. https://doi.org/10.1186/s12915-015-0184-7
Humphrey SJ, Azimifar SB, Mann M (2015) High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics. Nat Biotechnol 33:990–995. https://doi.org/10.1038/nbt.3327
Lasonder E, Treeck M, Alam M, Tobin AB (2012) Insights into the Plasmodium falciparum schizont phospho-proteome. Microbes Infect 14:811–819. https://doi.org/10.1016/j.micinf.2012.04.008
Leitner A (2016) Enrichment strategies in phosphoproteomics. In: Von Stecow L (ed) Methods in molecular biology. Springer, New York, pp 105–121
Dunn J, Reid G, Bruening ML (2010) Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry. Mass Spectrom Rev 29:29–54. https://doi.org/10.1002/mas
Mann M, Ong S, Gr M et al (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol 20:261–268. https://doi.org/10.1016/S0167-7799(02)01944-3
Olsen JV, Macek B, Lange O et al (2007) Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods 4:709–712. https://doi.org/10.1038/nmeth1060
FÃlla J, Honys D (2012) Enrichment techniques employed in phosphoproteomics. Amino Acids 43:1025–1047. https://doi.org/10.1007/s00726-011-1111-z
Engholm-Keller K, Birck P, Størling J et al (2012) TiSH — a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC. J Proteome 75:5749–5761. https://doi.org/10.1016/j.jprot.2012.08.007
Thingholm TE, Jensen ON, Robinson PJ, Larsen MR (2008) SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol Cell Proteomics 7:661–671. https://doi.org/10.1074/mcp.M700362-MCP200
Franck WL, Gokce E, Randall SM et al (2015) Phosphoproteome analysis links protein phosphorylation to cellular remodeling and metabolic adaptation during Magnaporthe oryzae appressorium development. J Proteome Res 14:2408–2424. https://doi.org/10.1021/pr501064q
Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11:2301–2319. https://doi.org/10.1038/nprot.2016.136
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906. https://doi.org/10.1038/nprot.2007.261
Acknowledgments
Support for this work was provided the National Science Foundation (MCB-0918611), the National Institute of Health Molecular Mycology and Pathogenesis Training program (5T32AI052080), and North Carolina State University to R.A.D.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Oh, Y., Franck, W.L., Dean, R.A. (2018). Sequential Phosphopeptide Enrichment for Phosphoproteome Analysis of Filamentous Fungi: A Test Case Using Magnaporthe oryzae. In: Ma, W., Wolpert, T. (eds) Plant Pathogenic Fungi and Oomycetes. Methods in Molecular Biology, vol 1848. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8724-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8724-5_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8723-8
Online ISBN: 978-1-4939-8724-5
eBook Packages: Springer Protocols