If the antibody fails -- a mass Western approach

Author(s)

Ute Lehmann, Stefanie Wienkoop, Hendrik Tschoep, Wolfram Weckwerth

Abstract

Sucrose-phosphate synthase (SPS) has attracted the interest of plant scientists for decades. It is the key

enzyme in sucrose metabolism and is under investigation in various plant species, e.g. spinach, tobacco,

poplar, resurrection plants, maize, rice, kiwi and Arabidopsis thaliana. In A. thaliana, there are four distinct SPS

isoforms. Their expression is thought to depend on environmental conditions and plant tissue. However, these

data were derived from mRNA expression levels only. No data on SPS protein identification from crude

extracts have been available until now. An antibody approach failed to distinguish the four isoforms.

Therefore, we developed a method for SPS quantification and isoform-specific identification in A. thaliana

complex protein samples. Samples were separated on SDS-PAGE, digested and directly applied to liquid

chromatography/triple-stage quadrupole mass spectrometry (LC/TSQ-MS). In this approach, known as mass

Western, samples were analysed in multi-reaction monitoring (MRM) mode, so that all four SPS isoforms could

be measured in one experiment. In addition to the relative quantification, stable isotope-labelled internal

peptide standards allowed absolute quantification of SPS proteins. Protein extracts from various plant tissues,

samples harvested during the day or the night, and cold-stressed plants were analysed. The stress-specific

SPS5a isoform showed increased concentrations in cold-stressed leaf material.

Organisation(s)

External organisation(s)

Universität Potsdam

Journal

The Plant Journal

Volume

Pages

1039-1046

No. of pages

ISSN

0960-7412

Publication date

2008

Peer reviewed

Yes

Austrian Fields of Science 2012

106002 Biochemistry, 1030 Physics, Astronomy, 106031 Plant physiology

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