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Titolo:
A practical protocol for the reduction of disulfide bonds in proteins prior to analysis by mass spectrometry
Autore:
Scigelova, M; Green, PS; Giannakopulos, AE; Rodger, A; Crout, DHG; Derrick, PJ;
Indirizzi:
Univ Warwick, Dept Chem, Coventry CV4 7AL, W Midlands, England Univ Warwick Coventry W Midlands England CV4 7AL 7AL, W Midlands, England Univ Warwick, Inst Mass Spectrometry, Coventry CV4 7AL, W Midlands, England Univ Warwick Coventry W Midlands England CV4 7AL 7AL, W Midlands, England
Titolo Testata:
EUROPEAN JOURNAL OF MASS SPECTROMETRY
fascicolo: 1, volume: 7, anno: 2001,
pagine: 29 - 34
SICI:
1469-0667(2001)7:1<29:APPFTR>2.0.ZU;2-8
Fonte:
ISI
Lingua:
ENG
Soggetto:
ELECTROSPRAY IONIZATION; CONFORMATIONS; LYSOZYME; SPECTRA; BRIDGES; INTACT;
Keywords:
protein; disulfide bond; mass spectrometry; circular dichroism; FTICR; electrospray ionisation;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Physical, Chemical & Earth Sciences
Citazioni:
11
Recensione:
Indirizzi per estratti:
Indirizzo: Giannakopulos, AE Univ Warwick, Dept Chem, Coventry CV4 7AL, W Midlands, England Univ Warwick Coventry W Midlands England CV4 7AL ngland
Citazione:
M. Scigelova et al., "A practical protocol for the reduction of disulfide bonds in proteins prior to analysis by mass spectrometry", EUR J MASS, 7(1), 2001, pp. 29-34

Abstract

A quick, simple applicable protocol for the reduction of protein disulfidebonds for the purposes of mass spectrometry has been established. The method utilises the chemical reducing agent dithiothreitol. Proteins with various numbers of disulfide bonds per molecule were chosen for the study to demonstrate the general applicability of the method. The results obtained under controlled conditions (concentration of reagents, pH, temperature) showedthat a five-minute treatment at 70 degreesC with 10 mM dithiothreitol in 5mM ammonium acetate buffer pH 5.5 was sufficient for complete reduction ofdisulfide bonds in all investigated proteins (alpha -lactalbumin, lysozyme, ribonuclease, oxytocin and wheat germ agglutinin). The progress of disulfide bond reduction was observed by electrospray ionisation and Fourier transform ion cyclotron resonance mass spectrometry. Circular dichroism was used to monitor conformational changes of reduced proteins and of their unreduced counterparts undergoing the same heat treatment.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 30/03/20 alle ore 00:41:26