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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

Miliotis, T.; Ericsson, P. O.; Marko-Varga, György LU ; Svensson, R.; Nilsson, J.; Laurell, Thomas LU and Bischoff, R. (2001) In Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences 752(2). p.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 was first 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.... (More)
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 was first 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 particular with respect to quantifying the status at the regulatory phosphorylation sites Tyr-492 and Tyr-493. Combination of chromatographic and on-line tandem mass 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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
phosphorylation, ZAP-70, PROTEIN-PHOSPHORYLATION, TRIFLUOROACETIC-ACID, COMPLEX-MIXTURES, TYROSINE KINASE, IDENTIFICATION, ACTIVATION, PEPTIDES, ONLINE
in
Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
volume
752
issue
2
pages
323 - 334
publisher
Elsevier
external identifiers
  • wos:000167347000012
  • scopus:0035836063
ISSN
0378-4347
DOI
10.1016/s0378-4347(00)00458-8
language
English
LU publication?
yes
id
f2ddb376-22dd-455e-b0af-e8a4f793fc81 (old id 2376400)
date added to LUP
2012-03-23 09:15:16
date last changed
2018-05-29 10:25:09
@article{f2ddb376-22dd-455e-b0af-e8a4f793fc81,
  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 was first 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 particular with respect to quantifying the status at the regulatory phosphorylation sites Tyr-492 and Tyr-493. Combination of chromatographic and on-line tandem mass 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.},
  author       = {Miliotis, T. and Ericsson, P. O. and Marko-Varga, György and Svensson, R. and Nilsson, J. and Laurell, Thomas and Bischoff, R.},
  issn         = {0378-4347},
  keyword      = {phosphorylation,ZAP-70,PROTEIN-PHOSPHORYLATION,TRIFLUOROACETIC-ACID,COMPLEX-MIXTURES,TYROSINE KINASE,IDENTIFICATION,ACTIVATION,PEPTIDES,ONLINE},
  language     = {eng},
  number       = {2},
  pages        = {323--334},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences},
  title        = {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},
  url          = {http://dx.doi.org/10.1016/s0378-4347(00)00458-8},
  volume       = {752},
  year         = {2001},
}