Issue 6, 2014

Fully automated on-line two-dimensional liquid chromatography in combination with ESI MS/MS detection for quantification of sugar phosphates in yeast cell extracts

Abstract

A mass spectrometric quantitative assay was developed for the analysis of 10 sugar phosphates in the yeast Pichia pastoris. As a novelty, two-dimensional chromatography based on a fully automated heart-cutting LC-LC technique was introduced. Using a ten-port valve, ten fractions of the first chromatographic dimension, i.e. anion exchange chromatography (AEC), were transferred and separated by the orthogonal second dimension, i.e. separation on porous graphitized carbon. The chromatographic separation on the second dimension was optimized for each transferred fraction minimizing the separation time and ensuring complete removal of the salt constituents of the AEC eluents. The latter being crucial for electrospray mass spectrometric detection was confirmed by combining the LC-LC separation with on-line ICP-MS detection. These measurements showed that sodium elution was completed after 0.8 min. Consequently, an analysis time of 1 min per transferred peak was established. In this way, the excellent peak capacity given by ion exchange could be conserved in the second dimension at the same time enabling mass spectrometric detection. Sub-μM limits of detection could be obtained by the new LC-LC-MS/MS methods ranging between 0.03 and 0.19 μM for the investigated compounds (only 3GAP showed a LOD of 1 μM). The method was applied to the quantification of ten sugar phosphates in yeast extracts utilizing internal standardization with a fully labeled 13C yeast extract. Typically, the standard uncertainties for N = 3 replicates assessed by the LC-LC-MS/MS set-up were <5%.

Graphical abstract: Fully automated on-line two-dimensional liquid chromatography in combination with ESI MS/MS detection for quantification of sugar phosphates in yeast cell extracts

Supplementary files

Article information

Article type
Paper
Submitted
11 Oct 2013
Accepted
08 Jan 2014
First published
08 Jan 2014

Analyst, 2014,139, 1512-1520

Fully automated on-line two-dimensional liquid chromatography in combination with ESI MS/MS detection for quantification of sugar phosphates in yeast cell extracts

K. Klavins, D. B. Chu, S. Hann and G. Koellensperger, Analyst, 2014, 139, 1512 DOI: 10.1039/C3AN01930F

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