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. 2009 Jul 28;2(81):pe45.
doi: 10.1126/scisignal.281pe45.

Proteomic revelation: SUMO changes partners when the heat is on

Karin Flick  1 , Peter Kaiser
Affiliations

Proteomic revelation: SUMO changes partners when the heat is on

Karin Flick et al. Sci Signal. .

Abstract

Dynamic changes in the posttranslational modification of proteins govern most cellular signaling pathways. Work over the past decade has connected many of these processes with the covalent attachment of the small ubiquitin-like modifier (SUMO) protein to target proteins, but a global view of the dynamics of SUMOylation was missing. A system-level proteomics approach has now been used to describe quantitative changes in protein modification with the SUMO-2 paralog during the response to heat shock. The SUMOylation status of more than 700 proteins was monitored in HeLa cells during the induction of hyperthermic stress and the recovery period. A massive redistribution of SUMO-2 was observed that affected many biological pathways that are important for the heat shock response, including cell cycle regulation, transcription, translation, protein folding, and DNA repair. Collectively, these data suggest a wide-ranging role for SUMOylation in the cellular response to hyperthermic stress. The strategies that were developed to provide this global view of SUMOylation should guide future approaches to probing quantitative changes in protein modification.

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Figures

Fig. 1
Fig. 1
Affinity purification and quantitative MS to identify dynamic changes in the SUMO-2 proteome during heat shock. SUMO-2 fused to an affinity-tag (TAPSUMO-2) was expressed in HeLa cells, and proteins modified with TAPSUMO-2 were separated from all other cell components by binding to affinity resins (20). Purified fractions were separated by SDS–polyacrylamide gel electrophoresis (PAGE) to reduce sample complexity before the MS analysis. Quantitative comparison of the SUMO-2 proteome from different samples was achieved by a triple-SILAC strategy (16), which is based on the metabolic incorporation of stable (nonradioactive) isotope–labeled amino acids into the proteome (–23). Three samples were compared: cells containing the TAP tag alone (for determination of purification background), cells containing TAPSUMO-2 that were incubated at 37°C, and cells containing TAPSUMO-2 that were incubated at 43°C for 2 hours. These different samples were cultured in “light,” “medium,” or “heavy” growth media, respectively. “Heavy” refers to the presence of Arg and Lys that were labeled with the stable isotopes 2H (deuterium), 13C, and 15N instead of the standard amino acids (1H, 12C, and 14N) in the “light” medium. The “medium” growth conditions contained Arg and Lys with an intermediate heavy isotope content. Heavy, medium, and light samples were analyzed together in the mass spectrometer, and identical peptides were detected as triplets because of the mass differences caused by the isotope-labeled amino acid residues. Relative quantitation was achieved by comparing the intensities of the peptide peaks. LC, liquid chromatography; m/z, mass/charge ratio.
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