Advanced

Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes

Hedström, Martin LU ; Grey, Carl LU ; Gaspar, Szilveszter LU and Mattiasson, Bo LU (2007) In Journal of Chromatography A 1146(1). p.17-22
Abstract
A miniaturized on-column digestion system constructed for the sequential analysis of semi-purified protein analytes is presented. By utilizing fused silica capillary (diameter 150 mu m) packed with a zone of trypsin-modified Eupergit C beads and a second zone of reversed-phase C18 material, a linear column set-up was constructed. The protein analytes (pmol amounts) were first digested in the 600 nl trypsin reactor portion of the system. Next, the generated peptides were trapped in the C18 column shaped as an electrospray emitter. Finally, after washing the matrix free from salts and other hydrophilic impurities present in the sample, peptides were eluted. A stepwise increased concentration profile of organic solvent, created by a dual... (More)
A miniaturized on-column digestion system constructed for the sequential analysis of semi-purified protein analytes is presented. By utilizing fused silica capillary (diameter 150 mu m) packed with a zone of trypsin-modified Eupergit C beads and a second zone of reversed-phase C18 material, a linear column set-up was constructed. The protein analytes (pmol amounts) were first digested in the 600 nl trypsin reactor portion of the system. Next, the generated peptides were trapped in the C18 column shaped as an electrospray emitter. Finally, after washing the matrix free from salts and other hydrophilic impurities present in the sample, peptides were eluted. A stepwise increased concentration profile of organic solvent, created by a dual syringe pump system, promoted the release of bound peptides, which were identified by electrospray ionization MS/MS. This approach proved to be very efficient, achieving almost complete digestion of the proteins studied, with suitable operational stability maintained for more than 1 week. Further, a small nebulizer was designed and fitted to the electrospray emitter. A significant improvement of the spray stability was observed and droplet build-up on the capillary was avoided, even at flow rates well above 1500 nl/min. The proteins chloroperoxidase, staphylococcal enterotoxin B and protein A (injection volume 0.3 mu l, salt concentration 0.2-1 M) were sequentially digested, desalted, eluted, detected and conclusively identified by bioinformatics web tools with an analytical cycle time of 10 min. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
on-line digestion, capillary, C18, protein, miniaturization, characterization
in
Journal of Chromatography A
volume
1146
issue
1
pages
17 - 22
publisher
Elsevier
external identifiers
  • wos:000245383200003
  • scopus:33847656700
ISSN
0021-9673
DOI
10.1016/j.chroma.2006.12.072
language
English
LU publication?
yes
id
33621f23-bb8f-4242-bd17-77f1a9b37551 (old id 668457)
date added to LUP
2007-12-11 13:35:47
date last changed
2017-01-01 07:10:06
@article{33621f23-bb8f-4242-bd17-77f1a9b37551,
  abstract     = {A miniaturized on-column digestion system constructed for the sequential analysis of semi-purified protein analytes is presented. By utilizing fused silica capillary (diameter 150 mu m) packed with a zone of trypsin-modified Eupergit C beads and a second zone of reversed-phase C18 material, a linear column set-up was constructed. The protein analytes (pmol amounts) were first digested in the 600 nl trypsin reactor portion of the system. Next, the generated peptides were trapped in the C18 column shaped as an electrospray emitter. Finally, after washing the matrix free from salts and other hydrophilic impurities present in the sample, peptides were eluted. A stepwise increased concentration profile of organic solvent, created by a dual syringe pump system, promoted the release of bound peptides, which were identified by electrospray ionization MS/MS. This approach proved to be very efficient, achieving almost complete digestion of the proteins studied, with suitable operational stability maintained for more than 1 week. Further, a small nebulizer was designed and fitted to the electrospray emitter. A significant improvement of the spray stability was observed and droplet build-up on the capillary was avoided, even at flow rates well above 1500 nl/min. The proteins chloroperoxidase, staphylococcal enterotoxin B and protein A (injection volume 0.3 mu l, salt concentration 0.2-1 M) were sequentially digested, desalted, eluted, detected and conclusively identified by bioinformatics web tools with an analytical cycle time of 10 min.},
  author       = {Hedström, Martin and Grey, Carl and Gaspar, Szilveszter and Mattiasson, Bo},
  issn         = {0021-9673},
  keyword      = {on-line digestion,capillary,C18,protein,miniaturization,characterization},
  language     = {eng},
  number       = {1},
  pages        = {17--22},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes},
  url          = {http://dx.doi.org/10.1016/j.chroma.2006.12.072},
  volume       = {1146},
  year         = {2007},
}