Abstract
In recent years, mass spectrometry-based phosphoproteomics has propelled our knowledge about the regulation of cellular pathways. Nevertheless, typically applied bottom-up strategies have several limitations. Trypsin, the preferentially used proteolytic enzyme shows impaired cleavage efficiency in the vicinity of phosphorylation sites. Moreover, depending on the frequency and distribution of tryptic cleavage sites (Arg/Lys), generated peptides can be either too short or too long for confident identification using standard LC-MS approaches. To overcome these limitations, we introduce an alternative and simple approach based on the usage of the nonspecific serine protease subtilisin, which enables a fast and reproducible digestion and provides access to “hidden” areas of the proteome. Thus, in a single LC-MS experiment >1800 phosphopeptides were confidently identified and localized from 125 μg of HeLa digest, compared to >2100 sites after tryptic digestion. While the overlap was less than 20 %, subtilisin allowed the identification of many phosphorylation sites that are theoretically not accessible via tryptic digestion, thus considerably increasing the coverage of the phosphoproteome.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Loroch S et al (2013) Phosphoproteomics—More than meets the eye. Electrophoresis 34(11):1483–1492
Mann M et al (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol 20(6):261–268
Yan JX et al (1998) Protein phosphorylation: technologies for the identification of phosphoamino acids. J Chromatogr A 808(1–2):23–41
Sickmann A, Meyer HE (2001) Phosphoamino acid analysis. Proteomics 1(2):200–206
Burkhart JM et al (2012) Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics. J Proteomics 75(4):1454–1462
Picotti P, Aebersold R, Domon B (2007) The Implications of Proteolytic Background for Shotgun Proteomics. Mol Cell Proteomics 6(9):1589–1598
Proc JL et al (2010) A Quantitative Study of the Effects of Chaotropic Agents, Surfactants, and Solvents on the Digestion Efficiency of Human Plasma Proteins by Trypsin. J Proteome Res 9(10):5422–5437
Olsen JV, Ong S-E, Mann M (2004) Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues. Mol Cell Proteomics 3(6):608–614
Bunkenborg J, Espadas G, Molina H (2013) Cutting Edge Proteomics: Benchmarking of Six Commercial Trypsins. J Proteome Res 12(8):3631–3641
Vandermarliere E, Mueller M, Martens L (2013) Getting intimate with trypsin, the leading protease in proteomics. Mass Spectrom Rev 32(6):453–465
Walmsley SJ et al (2013) Comprehensive Analysis of Protein Digestion Using Six Trypsins Reveals the Origin of Trypsin As a Significant Source of Variability in Proteomics. J Proteome Res 12(12):5666–5680
Dickhut C et al (2014) Impact of Digestion Conditions on Phosphoproteomics. J Proteome Res 13(6):2761–2770
Hamady M et al (2005) Does protein structure influence trypsin miscleavage? Engineering in Medicine and Biology Magazine. IEEE 24(3):58–66
Gilmore J, Kettenbach A, Gerber S (2012) Increasing phosphoproteomic coverage through sequential digestion by complementary proteases. Anal Bioanal Chem 402(2):711–720
Jacobs M et al (1985) Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res 13(24):8913–8926
Taus T et al (2011) Universal and Confident Phosphorylation Site Localization Using phosphoRS. J Proteome Res 10(12):5354–5362
Martens L, Vandekerckhove J, Gevaert K (2005) DBToolkit: processing protein databases for peptide-centric proteomics. Bioinformatics 21(17):3584–3585
Palmisano G et al (2012) A Novel Method for the Simultaneous Enrichment, Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development. Mol Cell Proteomics 11(11):1191–1202
Micallef L, Rodgers P (2014) eulerape: drawing area-proportional 3-Venn diagrams using ellipses. PLoS One 9(7):e101717
Acknowledgements
The financial support by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen and by the CAPES Foundation is gratefully acknowledged.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Gonczarowska-Jorge, H., Dell’Aica, M., Dickhut, C., Zahedi, R.P. (2016). Variable Digestion Strategies for Phosphoproteomics Analysis. In: von Stechow, L. (eds) Phospho-Proteomics. Methods in Molecular Biology, vol 1355. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3049-4_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3049-4_15
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4939-3048-7
Online ISBN: 978-1-4939-3049-4
eBook Packages: Springer Protocols