Chapter title |
Label-Free Identification and Quantification of SUMO Target Proteins.
|
---|---|
Chapter number | 13 |
Book title |
SUMO
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-6358-4_13 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6356-0, 978-1-4939-6358-4
|
Authors |
Ivo A. Hendriks, Alfred C. O. Vertegaal |
Editors |
Manuel S. Rodriguez |
Abstract |
Mass spectrometry-based approaches are utilized with increasing frequency to facilitate identification of novel SUMO target proteins and to elucidate the dynamics of SUMOylation in response to cellular stresses. Here, we describe a robust method for the identification of SUMO target proteins, and the relative quantification of SUMOylation dynamics, using a label-free approach. The method relies on a decahistidine (His10)-tagged SUMO, which is expressed at a low level in a mammalian cell line or model organism. The His10-tag allows for a single-step, high-yield, and high-purity enrichment of SUMOylated proteins, which are then digested and analyzed by high-resolution mass spectrometry. Matching between runs and label-free quantification integrated in the freely available MaxQuant software allow for a high rate and accuracy of quantification, providing a strong alternative to laborious sample or cell labeling techniques. The method described here allows for identification of >1000 SUMO target proteins, and characterization of their SUMOylation dynamics, without requiring sample fractionation. The purification procedure, starting from total lysate, can be performed in ~4 days. |
X Demographics
Geographical breakdown
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Unknown | 2 | 100% |
Demographic breakdown
Type | Count | As % |
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Members of the public | 2 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 33 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Student > Ph. D. Student | 9 | 27% |
Researcher | 5 | 15% |
Other | 3 | 9% |
Student > Bachelor | 3 | 9% |
Lecturer | 1 | 3% |
Other | 3 | 9% |
Unknown | 9 | 27% |
Readers by discipline | Count | As % |
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Biochemistry, Genetics and Molecular Biology | 17 | 52% |
Agricultural and Biological Sciences | 3 | 9% |
Immunology and Microbiology | 2 | 6% |
Unspecified | 1 | 3% |
Chemistry | 1 | 3% |
Other | 0 | 0% |
Unknown | 9 | 27% |