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.
Rapid Commun Mass Spectrom
; 27(1): 127-34, 2013 Jan 15.
Article
em En
| MEDLINE
| ID: mdl-23239325
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fragmentos de Peptídeos
/
Proteínas
/
Bases de Dados de Proteínas
/
Sumoilação
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Rapid Commun Mass Spectrom
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Reino Unido