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Titolo:
Characterization of phosphoproteins from electrophoretic gels by nanoscaleFe(III) affinity chromatography with off-line mass spectrometry analysis
Autore:
Stensballe, A; Andersen, S; Jensen, ON;
Indirizzi:
Odense Univ, Univ So Denmark, Dept Biochem & Mol Biol, DK-5230 Odense M, Denmark Odense Univ Odense Denmark M ochem & Mol Biol, DK-5230 Odense M, Denmark
Titolo Testata:
PROTEOMICS
fascicolo: 2, volume: 1, anno: 2001,
pagine: 207 - 222
SICI:
1615-9853(200102)1:2<207:COPFEG>2.0.ZU;2-P
Fonte:
ISI
Lingua:
ENG
Soggetto:
ELECTRON-CAPTURE DISSOCIATION; PROTEIN-KINASE-C; PHOSPHORYLATION SITES; SEQUENCE DATABASES; PEPTIDE MIXTURES; POSTTRANSLATIONAL MODIFICATIONS; IDENTIFYING PROTEINS; POLYACRYLAMIDE GELS; SELECTIVE DETECTION; 2-DIMENSIONAL GELS;
Keywords:
immobilized metal affinity chromatography phosphorylation; proteomics; mass spectrometry;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
69
Recensione:
Indirizzi per estratti:
Indirizzo: Jensen, ON Odense Univ, Univ So Denmark, Dept Biochem & Mol Biol, Campusvej 55, DK-5230 Odense M, Denmark Odense Univ Campusvej 55 Odense Denmark M 30 Odense M, Denmark
Citazione:
A. Stensballe et al., "Characterization of phosphoproteins from electrophoretic gels by nanoscaleFe(III) affinity chromatography with off-line mass spectrometry analysis", PROTEOMICS, 1(2), 2001, pp. 207-222

Abstract

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins is required to fully understand protein functionand regulatory events in cells and organisms. Here we present a mass spectrometry (MS) based experimental strategy for the identification and mappingof phosphorylation site(s) using only low-picomole amounts of phosphoprotein starting material. Miniaturized sample preparation methods for MS facilitated localization of phosphorylation sites in phosphoproteins isolated by polyacrylamide gel electrophoresis. Custom made, nanoscale immobilized Fe(III) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichment of phosphorylated peptides from crude peptide mixtures prior to off-line analysis by matrix-assisted laser desorption/ionization (MALDI) MS or nanoelectro-spray tandem mass spectrometry (MS/MS). An optimized and sensitive procedure for alkaline phosphatase treatment of peptide mixtures was implemented, which in combination with nano-scale Fe(lll)-IMAC and MALDI-MS allowed unambiguous identification of phosphopeptides by observation of 80 Da mass shifts. Nafioelectrospray MS/MS was used for phosphopeptide sequencingfor exact determination of phosphorylation sites. The advantages and limitations of the experimental strategy was demonstrated by enrichment, identification and sequencing of phosphopeptides from the model proteins ovalbuminand bovine beta -casein isolated by gel electrophoresis. Furthermore, an autophosphorylation site at Ser-3 in recombinant human casein kinase-2 beta subunit was determined. The potential of miniaturized Fe(lll)-IMAC and MALDI-MS for characterization of in vivo phosphorylated proteins was demonstrated by identification of tryptic phosphopeptides derived from the human p47/phox phosphoprotein isolated by two-dimensional gel electrophoresis.

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Documento generato il 30/03/20 alle ore 19:42:18