Abstract
Posttranslational modification (PTM) by the covalent conjugation of small ubiquitin-like modifier (SUMO) plays an important role in many biological processes, such as cell cycle progression, transcriptional regulation, subcellular transport, and other processes. An in-depth understanding of the function of SUMOylation requires the discovery of SUMO accepter sites. However, identification of endogenous SUMO-conjugated sites in higher eukaryotes by MS-based proteomic strategies is hampered by the low abundance of SUMO conjugates, the large tryptic fragments of SUMO1 or SUMO2/3 and the inability to match MS/MS spectra by protein database search engine. In this chapter, we describe a powerful method to overcome at least some of these challenges. To identify SUMO acceptor sites in endogenous SUMO1 conjugated protein, the SUMO1 conjugates are purified by immunoprecipitation with anti-SUMO1 antibodies followed by SDS-PAGE separation and in-gel tryptic digestion. The resulting peptides are either performed using standard data dependent acquisition (DDA) for protein identification or high mass DDA to enhance the sensitivity of detection on the LTQ-Orbitrap mass spectrometer. Finally, a Web-based database tool, ChopNSpice, coupled with a protein database search engine is introduced to ease the identification of SUMO1 attachment sites. Although this method was initially used to identify SUMO1 accepter sites, it can be readily adapted to study SUMO2/3 conjugates or even other Ubiquitin-like modifiers.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mann M, Ong SE, Gronborg M et al (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol 20:261–268
Kim SC, Sprung R, Chen Y et al (2006) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23:607–618
Meierhofer D, Wang X, Huang L, Kaiser P (2008) Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. J Proteome Res 7:4566–4576
Hsiao HH, Meulmeester E, Frank BT et al (2009) “ChopNSpice,” a mass spectrometric approach that allows identification of endogenous small ubiquitin-like modifier-conjugated peptides. Mol Cell Proteomics 8:2664–2675
Seeler JS, Dejean A (2003) Nuclear and unclear functions of SUMO. Nat Rev Mol Cell Biol 4:690–699
Johnson ES (2004) Protein modification by SUMO. Annu Rev Biochem 73:355–382
Hay RT (2005) SUMO: a history of modification. Mol Cell 18:1–12
Geiss-Friedlander R, Melchior F (2007) Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol 8:947–956
Meulmeester E, Melchior F (2008) Cell biology: SUMO. Nature 452:709–711
Panse VG, Hardeland U, Werner T et al (2004) A proteome-wide approach identifies sumoylated substrate proteins in yeast. J Biol Chem 279:41346–41351
Denison C, Rudner AD, Gerber SA et al (2005) A proteomic strategy for gaining insights into protein sumoylation in yeast. Mol Cell Proteomics 4:246–254
Wohlschlegel JA, Johnson ES, Reed SI, Yates JR 3rd (2004) Global analysis of protein sumoylation in Saccharomyces cerevisiae. J Biol Chem 279:45662–45668
Hannich JT, Lewis A, Kroetz MB et al (2005) Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae. J Biol Chem 280:4102–4110
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567
Eng JK, Mccormack AL, Yates JR (1994) An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database. J Am Soc Mass Spectrom 5:976–989
Olsen JV, de Godoy LM, Li G et al (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010–2021
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hsiao, HH., Meulmeester, E., Urlaub, H. (2012). Identification of Endogenous SUMO1 Accepter Sites by Mass Spectrometry. In: Marcus, K. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 893. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-885-6_27
Download citation
DOI: https://doi.org/10.1007/978-1-61779-885-6_27
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-884-9
Online ISBN: 978-1-61779-885-6
eBook Packages: Springer Protocols