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Titolo:
Toward efficient analysis of > 70 kDa proteins with 100% sequence coverage
Autore:
Forbes, AJ; Mazur, MT; Kelleher, NL; Patel, HM; Walsh, CT;
Indirizzi:
Univ Illinois, Dept Chem, Urbana, IL 61801 USA Univ Illinois Urbana IL USA 61801 linois, Dept Chem, Urbana, IL 61801 USA Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA Harvard Univ Boston MA USA 02115 em & Mol Pharmacol, Boston, MA 02115 USA
Titolo Testata:
EUROPEAN JOURNAL OF MASS SPECTROMETRY
fascicolo: 2, volume: 7, anno: 2001,
pagine: 81 - 87
SICI:
1469-0667(2001)7:2<81:TEAO>7>2.0.ZU;2-D
Fonte:
ISI
Lingua:
ENG
Soggetto:
RESOLUTION MASS-SPECTROMETRY; MULTIPLY-CHARGED IONS; ELECTROSPRAY-IONIZATION; LARGE MOLECULES; SPECTRA; EXPRESSION; GENOMICS; ACCURACY;
Keywords:
peptide mapping; limited proteolysis; electrospray; Fourier transform mass spectrometry;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Physical, Chemical & Earth Sciences
Citazioni:
28
Recensione:
Indirizzi per estratti:
Indirizzo: Kelleher, NL Univ Illinois, Dept Chem, 600 S Mathews Ave, Urbana, IL 61801USA Univ Illinois 600 S Mathews Ave Urbana IL USA 61801 61801 USA
Citazione:
A.J. Forbes et al., "Toward efficient analysis of > 70 kDa proteins with 100% sequence coverage", EUR J MASS, 7(2), 2001, pp. 81-87

Abstract

For complete characterization of larger proteins, primary structural analysis by mass spectrometry must be made more efficient. A straightforward approach is illustrated here using two proteins of 159 and 199 kDa with five and nine lysine (Lys) residues, respectively. These proteins were degraded by Lys-C to mixtures of peptides ranging in size from 5 to 48 kDa, whose multiply-charged ions (from electrospray ionization) are far more amenable than the intact proteins to direct interrogation in a Fourier-transform mass spectrometer. For the 199 kDa PchF of similar to 60% purity, an unfractionated Lys-C digest gave 106 isotopic distributions from 71 components (most ofwhich were below 6 kDa); 15% sequence coverage was obtained. For the > 90%pure PchE (159 kDa), complete sequence coverage was obtained from six Lys-C peptides of 5, 8, 26, 32, 40 and 48 kDa, with all but the largest of these measured at isotopic resolution on a 4.7 Tesla instrument. Practical strategies for implementing this characterization strategy on a proteomic scaleare considered.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 03/06/20 alle ore 09:44:19