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  Identification of endogenous SUMO1 accepter sites by mass spectrometry.

Hsiao, H. H., Meulmeester, E., & Urlaub, H. (2012). Identification of endogenous SUMO1 accepter sites by mass spectrometry. Quantitative methods in proteomics, 431-441. doi:10.1007/978-1-61779-885-6_27.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-000F-AAE3-7 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0029-1A78-2
Genre: Journal Article

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 Creators:
Hsiao, H. H.1, Author              
Meulmeester, E., Author
Urlaub, H.1, Author              
Affiliations:
1Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society, ou_578613              

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Free keywords: ChopNSpice - Liquid chromatography - Mass spectrometry - Posttranslational modification - Proteomics - Small ubiquitin-like modifier
 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.

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Language(s): eng - English
 Dates: 2012-06-302012
 Publication Status: Published in print
 Pages: -
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 Rev. Method: -
 Identifiers: DOI: 10.1007/978-1-61779-885-6_27
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Title: Quantitative methods in proteomics
Source Genre: Book
 Creator(s):
Marcus, K., Editor
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Publ. Info: New York : Humana Pr.; Springer
Pages: XV, 539 Volume / Issue: - Sequence Number: - Start / End Page: 431 - 441 Identifier: ISBN: 978-1-61779-884-9

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Title: Methods in molecular biology
Source Genre: Series
 Creator(s):
Walker, J. M., Editor
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Pages: - Volume / Issue: 893 Sequence Number: - Start / End Page: - Identifier: -