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If the antibody fails--a mass western approach.

Lehmann U, Wienkoop S, Tschoep H, Weckwerth W - Plant J. (2008)

Bottom Line: An antibody approach failed to distinguish the four isoforms.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.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Plant Physiology, Am M├╝hlenberg 1, 14424 Potsdam, Germany.

ABSTRACT

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.

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SPS quantification in various A. thaliana tissues.(a) SPS quantification in ammonium sulfate precipitates of proteins extracted from cold-stressed leaves. Ammonium sulfate precipitates (30, 40 and 50%) were taken up in native buffer and aliquots applied to SDS-PAGE. The amount of each SPS isoform (in pmol) detected in the 120 kDa gel band is shown.(b) SPS quantification in ammonium sulfate precipitates of extracted flower proteins. Ammonium sulfate precipitates (30, 40 and 50%) were taken up in native buffer and aliquots applied to SDS-PAGE. The amount of each SPS isoform (in pmol) detected in the 120 kDa band is shown.
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fig03: SPS quantification in various A. thaliana tissues.(a) SPS quantification in ammonium sulfate precipitates of proteins extracted from cold-stressed leaves. Ammonium sulfate precipitates (30, 40 and 50%) were taken up in native buffer and aliquots applied to SDS-PAGE. The amount of each SPS isoform (in pmol) detected in the 120 kDa gel band is shown.(b) SPS quantification in ammonium sulfate precipitates of extracted flower proteins. Ammonium sulfate precipitates (30, 40 and 50%) were taken up in native buffer and aliquots applied to SDS-PAGE. The amount of each SPS isoform (in pmol) detected in the 120 kDa band is shown.

Mentions: For both cold-stressed leaf and flower samples, the biological replicates showed high standard deviations (see Figure 3a,b). However, in these protein fractions extracted from A. thaliana, clear trends indicating that SPS5a is the dominant isoform in cold-stressed leaves and SPS1 is the dominant isoform in flowers were observed (Figure 3a,b).


If the antibody fails--a mass western approach.

Lehmann U, Wienkoop S, Tschoep H, Weckwerth W - Plant J. (2008)

SPS quantification in various A. thaliana tissues.(a) SPS quantification in ammonium sulfate precipitates of proteins extracted from cold-stressed leaves. Ammonium sulfate precipitates (30, 40 and 50%) were taken up in native buffer and aliquots applied to SDS-PAGE. The amount of each SPS isoform (in pmol) detected in the 120 kDa gel band is shown.(b) SPS quantification in ammonium sulfate precipitates of extracted flower proteins. Ammonium sulfate precipitates (30, 40 and 50%) were taken up in native buffer and aliquots applied to SDS-PAGE. The amount of each SPS isoform (in pmol) detected in the 120 kDa band is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2607522&req=5

fig03: SPS quantification in various A. thaliana tissues.(a) SPS quantification in ammonium sulfate precipitates of proteins extracted from cold-stressed leaves. Ammonium sulfate precipitates (30, 40 and 50%) were taken up in native buffer and aliquots applied to SDS-PAGE. The amount of each SPS isoform (in pmol) detected in the 120 kDa gel band is shown.(b) SPS quantification in ammonium sulfate precipitates of extracted flower proteins. Ammonium sulfate precipitates (30, 40 and 50%) were taken up in native buffer and aliquots applied to SDS-PAGE. The amount of each SPS isoform (in pmol) detected in the 120 kDa band is shown.
Mentions: For both cold-stressed leaf and flower samples, the biological replicates showed high standard deviations (see Figure 3a,b). However, in these protein fractions extracted from A. thaliana, clear trends indicating that SPS5a is the dominant isoform in cold-stressed leaves and SPS1 is the dominant isoform in flowers were observed (Figure 3a,b).

Bottom Line: An antibody approach failed to distinguish the four isoforms.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.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Plant Physiology, Am M├╝hlenberg 1, 14424 Potsdam, Germany.

ABSTRACT

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.

Show MeSH