Abstract
Mass spectrometry (MS) is a sensitive analytical technique with wide application across the sciences including for the detection of peptides and proteins in biological analysis. Ubiquitinated (Ub) proteins are typically analyzed by proteolytic digestion and subsequent chromatographic separation followed by MS detection of the resulting isopeptides. Here we describe a novel method which enables enhanced detection of this important posttranslational modification (PTM) by use of a simple chemical labeling strategy prior to Data-Independent Acquisition (DIA) using a SWATH-based acquisition approach on a suitable Quadrupole-Time-Of-Flight (Q-TOF) mass spectrometer.
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
Finley D, Chau V (1991) Ubiquitination. Annu Rev Cell Biol 7:25–69
Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315:201–205
Bergink S, Jentsch S (2009) Principles of ubiquitin and SUMO modifications in DNA repair. Nature 458:461–467
Hochstrasser M (2009) Origin and function of ubiquitin-like proteins. Nature 458:422–429
Sun Y (2006) E3 ubiquitin ligases as cancer targets and biomarkers. Neoplasia 8:645–654
Bologna S, Ferrari S (2013) It takes two to tango: ubiquitin and SUMO in the DNA damage response. Front Genet 4:1–18
Chen Z, Lu W (2015) Roles of ubiquitination and SUMOylation on prostate cancer: mechanisms and clinical implications. Int J Mol Sci 16:4560–4580
Peng J, Schwartz D, Elias JE et al (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21(8):921–926
Nielsen ML, Vermeulen M, Bonaldi T et al (2008) Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat Methods 5:459–460
Warren MRE, Parker CE, Mocanu V et al (2005) Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic product ions for protein ubiquitination. Rapid Commun Mass Spectrom 19:429–437
Chicooree N, Connolly Y, Tan C-T et al (2013) Enhanced detection of isopeptides using reductive methylation. J Am Soc Mass Spectrom 24:421–430
Fu Q, Li L (2006) Fragmentation of peptides with N-terminal dimethylation and imine/methylol adduction at the tryptophan side-chain. J Am Soc Mass Spectrom 17:859–866
Hsu JL, Huang SY, Chow NH et al (2003) Stable-isotope dimethyl labeling for quantitative proteomics. Anal Chem 75:6843–6852
Gillet LC, Navarro P, Tate S et al (2012) Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics 11:O111.016717
Griffiths JR, Chicooree N, Connolly Y et al (2014) Mass spectral enhanced detection of Ubls using SWATH acquisition: MEDUSA—simultaneous quantification of SUMO and ubiquitin-derived isopeptides. J Am Soc Mass Spectrom 25:767–777
Chicooree N, Smith DL (2016) Mass spectrometry methods for the analysis of isopeptides generated from mammalian protein ubiquitination and SUMOylation. In: Griffiths JR, Unwin RD (eds) Analysis of protein post-translational modifications by mass spectrometry. John Wiley and Sons, Hoboken, NJ
https://sciex.com/Documents/Downloads/Literature/mass-spectrometry-4373841D.pdf
Perkins DN, Pappin DJ, Creasy DM et al (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567
MacLean B, Tomazela DM, Shulman N et al (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26:966–968
Acknowledgments
We would like to acknowledge our coauthors on the original paper [15], for their equal contribution to the development of the MEDUSA workflow described herein.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Chicooree, N., Griffiths, J.R. (2019). A Combined Chemical Derivatization/Mass Spectrometric Method for the Enhanced Detection and Relative Quantification of Protein Ubiquitination. In: Evans, C., Wright, P., Noirel, J. (eds) Mass Spectrometry of Proteins. Methods in Molecular Biology, vol 1977. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9232-4_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9232-4_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9231-7
Online ISBN: 978-1-4939-9232-4
eBook Packages: Springer Protocols