Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes
(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:
https://lup.lub.lu.se/record/668457
- author
- Hedström, Martin LU ; Grey, Carl LU ; Gaspar, Szilveszter LU and Mattiasson, Bo LU
- organization
- publishing date
- 2007
- 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
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Biotechnology (LTH) (011001037), Analytical Chemistry (S/LTH) (011001004)
- id
- 33621f23-bb8f-4242-bd17-77f1a9b37551 (old id 668457)
- date added to LUP
- 2016-04-01 16:36:50
- date last changed
- 2022-01-28 20:55:54
@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}},
keywords = {{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}},
doi = {{10.1016/j.chroma.2006.12.072}},
volume = {{1146}},
year = {{2007}},
}