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Titolo:
RAPID MICROSCALE PROTEOLYSIS OF PROTEINS FOR MALDI-MS PEPTIDE-MAPPINGUSING IMMOBILIZED TRYPSIN
Autore:
GOBOM J; NORDHOFF E; EKMAN R; ROEPSTORFF P;
Indirizzi:
ODENSE UNIV,DEPT MOL BIOL,CAMPUSVEJ 55 DK-5230 ODENSE M DENMARK ODENSE UNIV,DEPT MOL BIOL DK-5230 ODENSE M DENMARK GOTHENBURG UNIV,MOELNDAL HOSP,DEPT CLIN NEUROSCI,SECT PSYCHIAT & NEUROCHEM S-43180 MOELNDAL SWEDEN
Titolo Testata:
International journal of mass spectrometry and ion processes
, volume: 169, anno: 1997,
pagine: 153 - 163
SICI:
0168-1176(1997)169:<153:RMPOPF>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
SEQUENCE DATABASES; MASS; IDENTIFICATION;
Keywords:
IMMOBILIZED PROTEASES; MALDI-MS; PEPTIDE MAPPING; PROTEIN IDENTIFICATION; TRYPTIC DIGESTION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
15
Recensione:
Indirizzi per estratti:
Citazione:
J. Gobom et al., "RAPID MICROSCALE PROTEOLYSIS OF PROTEINS FOR MALDI-MS PEPTIDE-MAPPINGUSING IMMOBILIZED TRYPSIN", International journal of mass spectrometry and ion processes, 169, 1997, pp. 153-163

Abstract

In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution-and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution ofthe digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversed-phase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion. (C) 1997 Elsevier Science B.V.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 04/12/20 alle ore 04:03:00