Advanced

Interfacing HPLC with MALDI- and ESI-MS for Automated High Sensitivity Analysis of Proteins and Peptides

Miliotis, Tasso LU (2001)
Abstract
The research described in this thesis has an interdisciplinary approach dealing with chemical, biological, and technical issues. One objective was to interface miniaturized liquid chromatography (LC) to mass spectrometry, i.e. MALDI-TOF MS and ESI-MS, in an unattended mode for high sensitivity determination of proteins and peptides. The coupling between capillary LC and MALDI was accomplished by employing a piezoelectric flow-through microdispenser. The column effluent was nano fractionated onto a MALDI target plate with a negligible dispersion, thus transferring the high-resolution separation from the LC system onto the target plate as discrete spots. In conjunction, a simple sample preparation technique was developed that facilitated... (More)
The research described in this thesis has an interdisciplinary approach dealing with chemical, biological, and technical issues. One objective was to interface miniaturized liquid chromatography (LC) to mass spectrometry, i.e. MALDI-TOF MS and ESI-MS, in an unattended mode for high sensitivity determination of proteins and peptides. The coupling between capillary LC and MALDI was accomplished by employing a piezoelectric flow-through microdispenser. The column effluent was nano fractionated onto a MALDI target plate with a negligible dispersion, thus transferring the high-resolution separation from the LC system onto the target plate as discrete spots. In conjunction, a simple sample preparation technique was developed that facilitated automated and unattended MALDI-TOF MS. A methodology for rapid analysis of phosphorylation sites utilizing capillary LC-ESI/MS/MS is presented. The combination of microcolumn chromatography and tandem mass spectrometry was employed for differentiation of the phosphorylation status of the tyrosine kinase ZAP-70. An automated comprehensive two-dimensional (2D) chromatography system was developed for the mapping of proteins below 30 kDa. In addition, a sample preparation step using restricted access materials (RAMs) was built in-line prior the first dimension, enabling selective sample enrichment. Biological samples, e.g. human hemofiltrate, were used to demonstrate the high resolving power of the 2D-LC system. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Irth, Hubertus
organization
publishing date
type
Thesis
publication status
published
subject
keywords
automated, phosphorylation, ZAP-70, peptides, proteins, comprehensive, multidimensional, two-dimensional, piezoelectric, microdispenser, flow-through, ESI-MS/MS, Capillary HPLC, MALDI-TOF MS, Analytical chemistry, Analytisk kemi
pages
176 pages
publisher
Department of Analytical Chemistry, Lund University
defense location
Lecture Hall B, Chemical Center
defense date
2000-03-30 10:15
external identifiers
  • Other:ISRN: LUNKDL/NKAK-1057/1-170 (2001)
ISBN
91-7874-124-6
language
English
LU publication?
yes
id
05ccbfc8-7dd0-48e4-b4cf-9e62fc29c08c (old id 41403)
date added to LUP
2007-08-01 14:58:47
date last changed
2016-09-19 08:45:09
@phdthesis{05ccbfc8-7dd0-48e4-b4cf-9e62fc29c08c,
  abstract     = {The research described in this thesis has an interdisciplinary approach dealing with chemical, biological, and technical issues. One objective was to interface miniaturized liquid chromatography (LC) to mass spectrometry, i.e. MALDI-TOF MS and ESI-MS, in an unattended mode for high sensitivity determination of proteins and peptides. The coupling between capillary LC and MALDI was accomplished by employing a piezoelectric flow-through microdispenser. The column effluent was nano fractionated onto a MALDI target plate with a negligible dispersion, thus transferring the high-resolution separation from the LC system onto the target plate as discrete spots. In conjunction, a simple sample preparation technique was developed that facilitated automated and unattended MALDI-TOF MS. A methodology for rapid analysis of phosphorylation sites utilizing capillary LC-ESI/MS/MS is presented. The combination of microcolumn chromatography and tandem mass spectrometry was employed for differentiation of the phosphorylation status of the tyrosine kinase ZAP-70. An automated comprehensive two-dimensional (2D) chromatography system was developed for the mapping of proteins below 30 kDa. In addition, a sample preparation step using restricted access materials (RAMs) was built in-line prior the first dimension, enabling selective sample enrichment. Biological samples, e.g. human hemofiltrate, were used to demonstrate the high resolving power of the 2D-LC system.},
  author       = {Miliotis, Tasso},
  isbn         = {91-7874-124-6},
  keyword      = {automated,phosphorylation,ZAP-70,peptides,proteins,comprehensive,multidimensional,two-dimensional,piezoelectric,microdispenser,flow-through,ESI-MS/MS,Capillary HPLC,MALDI-TOF MS,Analytical chemistry,Analytisk kemi},
  language     = {eng},
  pages        = {176},
  publisher    = {Department of Analytical Chemistry, Lund University},
  school       = {Lund University},
  title        = {Interfacing HPLC with MALDI- and ESI-MS for Automated High Sensitivity Analysis of Proteins and Peptides},
  year         = {2001},
}