If the antibody fails – a mass Western approach

SUMMARY 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.