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Titolo:
Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Autore:
Miliotis, T; Ericsson, PO; Marko-Varga, G; Svensson, R; Nilsson, J; Laurell, T; Bischoff, R;
Indirizzi:
AstraZeneca, R&D Lund, Dept Mol Sci, SE-22187 Lund, Sweden AstraZeneca Lund Sweden SE-22187 nd, Dept Mol Sci, SE-22187 Lund, Sweden Univ Lund, Dept Analyt Chem, SE-22100 Lund, Sweden Univ Lund Lund SwedenSE-22100 , Dept Analyt Chem, SE-22100 Lund, Sweden AstraZeneca Biotech Lab, SE-15185 Sodertalje, Sweden AstraZeneca Biotech Lab Sodertalje Sweden SE-15185 85 Sodertalje, Sweden Lund Inst Technol, Dept Elect Measurements, SE-22100 Lund, Sweden Lund Inst Technol Lund Sweden SE-22100 asurements, SE-22100 Lund, Sweden
Titolo Testata:
JOURNAL OF CHROMATOGRAPHY B
fascicolo: 2, volume: 752, anno: 2001,
pagine: 323 - 334
SICI:
1387-2273(20010310)752:2<323:AORPSI>2.0.ZU;2-D
Fonte:
ISI
Lingua:
ENG
Soggetto:
PROTEIN-PHOSPHORYLATION; TRIFLUOROACETIC-ACID; COMPLEX-MIXTURES; TYROSINE KINASE; IDENTIFICATION; ACTIVATION; PEPTIDES; ONLINE;
Keywords:
phosphorylation; ZAP-70;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
24
Recensione:
Indirizzi per estratti:
Indirizzo: Bischoff, R AstraZeneca, R&D Lund, Dept Mol Sci, Scheelevagen 8, SE-22187 Lund, Sweden AstraZeneca Scheelevagen 8 Lund Sweden SE-22187 Lund, Sweden
Citazione:
T. Miliotis et al., "Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry", J CHROMAT B, 752(2), 2001, pp. 323-334

Abstract

A methodology for the rapid and quantitative analysis of phosphorylation sites in proteins is presented. The coupling of capillary high-performance liquid chromatography (HPLC) to electrospray ionization mass spectrometry (ESI-MS) allowed one to distinguish phosphorylation sites based on retention time and mass difference from complex peptide mixtures. The methodology wasfirst evaluated and validated for a mixture of non-, mono-, and dityrosine-phosphorylated synthetic peptides, corresponding to the tryptic fragment 485-496 (ALGADDSYYTAR) of the human protein tyrosine kinase ZAP-70. The limits of detection for the non-, mono- and diphosphorylated peptides were about 15. 40 and 100 fmol, respectively, when using a 300 mum I.D. column. Application of the method was extended to identify phosphopeptides generated from a trypsin digest of recombinant autophosphorylated ZAP-70, in particularwith respect to quantifying the status at the regulatory phosphorylation sites Tyr-492 and Tyr-493. Combination of chromatographic and on-line tandemmass spectrometry data, allowed one to ascertain the identity of the detected peptides, a prerequisite to analyses in more complex biological samples. As an extension to the methodology described above, we evaluated the feasibility of interfacing capillary HPLC to matrix assisted laser desorption ionisation time-of-Right mass spectrometry (MALDI-TOF-MS), using a micromachined piezoelectric flow-through dispenser as the interface. This enabled direct arraying of chromatographically separated components onto a target plate that was precoated with matrix for subsequent analysis by MALDI-TOF-MS without further sample handling. (C) 2001 Elsevier Science B.V. All rights reserved.

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Documento generato il 04/04/20 alle ore 01:21:24