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Pillar array columns for peptide separations in nanoscale reversed-phase chromatography.

Authors
  • Tóth, Gábor1
  • Panić-Janković, Tanja2
  • Mitulović, Goran3
  • 1 Proteomics Core Facility, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria; Budapest University of Technology and Economics, Faculty of Chemical Technology and Biotechnology, Műegyetem rkp 3, 1111 Budapest, Hungary; MS Proteomics Research Group, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt 2, 1117 Budapest, Hungary. , (Austria)
  • 2 Clinical Department of Laboratory Medicine, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. , (Austria)
  • 3 Proteomics Core Facility, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria; Clinical Department of Laboratory Medicine, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. Electronic address: [email protected] , (Austria)
Type
Published Article
Journal
Journal of chromatography. A
Publication Date
Oct 11, 2019
Volume
1603
Pages
426–432
Identifiers
DOI: 10.1016/j.chroma.2019.06.067
PMID: 31301798
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Reversed-phase chromatography is the most common technique for separation of tryptic peptides. In this short communication, we describe the optimization of sample loading and separation parameters for a novel micromachined column and provide a detailed description on the performance and reproducibility of this separation system. Tryptic digest of a mixture of seven proteins with diverse mass and isoelectric point was used as a test sample. The methods developed and used are straight-forward; by using well-balanced, combined-step gradients an optimal distribution of peptides on the column could be achieved throughout the complete gradient window. The potential use of the column is exceptional due to the low back-pressure, better distribution of peptides over the separation window, enhanced stability and reproducibility of retention times, and the prolonged lifetime of columns compared to conventionally packed nano-HPLC column. The higher identification rates have been demonstrated through measurements of HeLa cell lysates under identical chromatographic conditions on the pillar array and packed-bed columns. Copyright © 2019 Elsevier B.V. All rights reserved.

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