Abstract
Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides. To overcome these problems, we created HEK293 cell lines that stably express functional SUMO paralogs with an N-terminal His6-tag and an Arg residue near the C terminus that leave a short five amino acid SUMO remnant upon tryptic digestion. We determined the fragmentation patterns of our short SUMO remnant peptides by collisional activation and electron transfer dissociation using synthetic peptide libraries. Activation using higher energy collisional dissociation on the LTQ-Orbitrap Elite identified SUMO paralog-specific fragment ions and neutral losses of the SUMO remnant with high mass accuracy (< 5 ppm). We exploited these features to detect SUMO modified tryptic peptides in complex cell extracts by correlating mass measurements of precursor and fragment ions using a data independent acquisition method. We also generated bioinformatics tools to retrieve MS/MS spectra containing characteristic fragment ions to the identification of SUMOylated peptide by conventional Mascot database searches. In HEK293 cell extracts, this MS approach uncovered low abundance SUMOylated peptides and 37 SUMO3-modified Lys residues in target proteins, most of which were previously unknown. Interestingly, we identified mixed SUMO-ubiquitin chains with ubiquitylated SUMO proteins (K20 and K32) and SUMOylated ubiquitin (K63), suggesting a complex crosstalk between these two modifications.
Highlights
From the ‡Institute for Research in Immunology and Cancer, §Department of Chemistry, §§Department of Biochemistry, Universitede Montreal, P.O
Product Ion Spectra of small ubiquitin-like modifier (SUMO) Mutant Peptides Revealed Paralog-specific Fragment Ions—The identification of SUMOylated peptides by conventional database search engines can be challenging in view of the occurrence of fragment ions from the peptide backbone and the long SUMO remnant chain that complicates the interpretation of the corresponding mass spectrometry (MS)/MS spectra [16, 17]
Synthetic peptides containing either a SUMO1 (EQTGG) or SUMO3 (NQTGG) remnant chain were synthesized on solid phase support and were selected based on sequences predicted from the tryptic digestion of known SUMOylated proteins
Summary
Peptide Synthesis—The peptides [1] Boc-Glu(OtBu)-Qln(Trt)Thr(tBu)-Gly-Gly-OH and [2] Boc-Asn(Trt)-Gln(Trt)-Thr(tBu)-GlyGly-OH were synthesized manually on a 2-chlorotrityl chloride resin using standard procedures of solid phase peptide synthesis. Protein Digestion—SUMO-proteins immobilized on NTA beads were solubilized in 4 M urea, reduced in 5 mM tris(2-carboxyethyl)phosphine (TCEP) (Pierce) for 20 min at 37 °C and alkylated in 5 mM chloroacetamide (Sigma-Aldrich) for 20 min at 37 °C We used this alkylating agent to differentiate free cysteines (modified by NEM) and residues linked by disulfide-bonds (modified by chloroacetamide). An injection time of 50 ms for a target value of 106 counts was used for the HCD MS/MS acquisition This experiment enabled the generation of an inclusion list of potential SUMO peptides for subsequent targeted MS/MS experiments. A software application was developed to search Mascot generic files (mgf) for specific SUMO fragment ions (e.g. SUMO1: m/z 102.0550, 129.0659, 240.0979, 258.1084, 341.1456, 359.1561; SUMO3: m/z 132.0768, 226.0822, 243.1088, 299.1350, 316.1615, 326.1459, 327.1299, 344.1565, 383.1674, 401.1779; and neutral losses of SUMO remnants) to produce a subset mgf file containing only MS/MS spectra of potential SUMOylated peptide candidates.
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