Premium
A novel approach to the analysis of SUMOylation with the independent use of trypsin and elastase digestion followed by database searching utilising consecutive residue addition to lysine
Author(s) -
Chicooree Navin,
Griffiths John R.,
Connolly Yvonne,
Tan ChongTeik,
Malliri Angeliki,
Eyers Claire E.,
Smith Duncan L.
Publication year - 2012
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6425
Subject(s) - sumo protein , trypsin , orbitrap , chemistry , electrospray ionization , sumo enzymes , mass spectrometry , database , lysine , tandem mass spectrometry , computational biology , chromatography , biochemistry , enzyme , biology , computer science , ubiquitin , amino acid , gene
RATIONALE Identification of sites of protein SUMOylation is of great importance due its functional diversity within the cell. To date, most approaches to this problem rely on site‐directed mutagenesis and/or highly specialised mass spectrometry approaches. We present a novel alternative approach to the site mapping of SUMOylation using trypsin and elastase digestion, routine mass spectrometry and an unbiased isotag database searching strategy. METHODS SUMOylated protein samples were digested with a number of enzymes and the resulting peptides separated using liquid chromatography. Analysis was carried out on both linear ion trap Orbitrap and quadrupole‐time‐of‐flight (Q‐TOF)‐based mass spectrometers equipped with electrospray ionisation. The data files were subsequently searched using the Mascot algorithm with multiple variable tag modifications corresponding to SUMO‐derived fragments. The utility of this approach was demonstrated with di‐SUMO 2, di‐SUMO 3, SUMO 1‐RanGap 418‐587 1 and an enriched population of SUMOylated proteins. RESULTS Unbiased database searches led to the identification of a number of analytically useful isotags ranging in length from two to four residues. Isopeptide fragments were generated including QTGG (di‐SUMO‐2/3), TGG (di‐SUMO‐2/3) and GG (SUMO‐1). The method was validated by successfully mapping a number of sites of SUMO modification on SUMO‐modified proteins enriched from a cell lysate. CONCLUSIONS This combination of relaxed enzyme specificity, shortened isotag generation and unbiased database searching enabled confident identification of novel analytically useful SUMOylated isopeptides without a requirement for mutagenesis. Copyright © 2012 John Wiley & Sons, Ltd.