title: 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.
abstract: BACKGROUND
  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.
authors.affiliation: Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK.
authors.forename: Navin
authors.initials: N
authors.lastname: Chicooree
authors.forename: John R
authors.initials: JR
authors.lastname: Griffiths
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Connolly
authors.forename: Chong-Teik
authors.initials: CT
authors.lastname: Tan
authors.forename: Angeliki
authors.initials: A
authors.lastname: Malliri
authors.forename: Claire E
authors.initials: CE
authors.lastname: Eyers
authors.forename: Duncan L
authors.initials: DL
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D019295:Computational Biology; D030562:Databases, Protein; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D058207:Sumoylation; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D010196:Pancreatic Elastase; D014357:Trypsin; D008239:Lysine