Abstract
The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His(6) tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation.
Highlights
The small ubiquitin-like modifier (SUMO)1 proteins are structurally similar to ubiquitin, they share less than
The short five-amino acid segment appended to the modified Lys residues (e.g. EQTGG for SUMO1 mut; Fig. 1a) result in fewer fragment ions from the Lys side chain, a property that favors the identification of SUMOylated peptides using conventional database search engines
Plasmids were constructed with single base substitutions at the C-terminal end of SUMO paralogs to generate mature proteins with an Arg residue at the sixth position from the C terminus similar to that found in the yeast Smt3 protein
Summary
Plasmid Construction and Generation of Stable HEK293 Cells Expressing SUMO Constructs—cDNAs of His6-SUMO wild type (WT) and His6-SUMO mutants were generated by PCR with the forward primer (5Ј-gacccaagcttggtaccatggctcatc-3Ј) containing the His tag and KpnI and NcoI restriction sites and the reverse primers containing the stop codon and XhoI restriction site. Enrichment of SUMOylated Proteins—Cells stably expressing His6SUMO (107 cells) were lysed in denaturing buffer A (6 M guanidinium HCl, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8, 10 mM -mercaptoethanol), sonicated, centrifuged at 16,000 ϫ g, and incubated with 50 l of NTA-agarose beads (Invitrogen) for 3 h [31]. Confocal Microscopy—HEK293 cells, transfected with pcDNA3His6-SUMO and pcDNA3-PMLIII-YFP, were fixed in 4% paraformaldehyde for 10 min at 4 °C and revealed by anti-His antibody followed by Alexa Fluor 594-conjugated antibody. We developed a Perl script that searched the Mascot generic file (mgf) for the specific SUMO fragment ions (e.g. SUMO1: m/z 240.1, 258.1, 341.1, and 359.2; SUMO3: m/z 243.1 and 344.2; and neutral losses of SUMO remnants) to produce a subset mgf file containing all MS/MS spectra of potential SUMOylated peptide candidates. Manual inspection of all MS/MS spectra for modified peptides was performed to validate assignments (supplemental Fig. S8)
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