Semi-targeted UPLC -MS analysis of phenylpropanoids in Arabidopsis
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Transcript of Semi-targeted UPLC -MS analysis of phenylpropanoids in Arabidopsis
Why phenylpropanoids?
- they may be involved in cell proliferation and differentiation (Tamagnone et al., 1998; Tsukagoshi et al., 2011)
- they are induced by cytokinins (Deikman et al., 1995)
Semi-targeted analysis (vs. targeted)
Pros
- fast analysis of all detectable metabolites in a simple run
- effective selection of metabolites which account for the most
variability in the dataset
- most suitable for abundant compounds, such as
phenylpropanoids
Cons
- low sensitivity with many instrumentations
- complicated use of internal standards
- more technical replicates needed (pooled samples), lower column
lifetime
- challenging data processing
Semi-targeted UPLC analysis
Biological replicates
Pooled samples
Extraction
Randomiz., UPLC-MS
Data filtering,chromatogram building
& deconvolutionpeak detection & alignment
Raw data export
Data export
Statistical software,multivariate analysis (MVA)
CRITICAL!
Differential analysis of WT vs. pal1 by
UPLC-MS
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5
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WT pal2 pal1
*
***
OR
AC
(μ
mol
trol
ox e
quiv
alen
ts g
FW
)-1
A) B)
Fig. 1 (A) Flowering phenotypes of wild-type and pal mutant plants of Arabidopsis thaliana. (B) Oxygen radical absorbance capacity (ORAC) of wild-type and pal mutant plants. Bars represent the means (±SE) of ten individual plants. Asterisks indicate significant differences between wild-type (WT) and pal-mutants as determined by Student’s t test (* P<0.05, *** P<0.001).
Flowering phenotypes of Col wild-type (WT) and pal mutant plants of Arabidopsis thaliana grown at low density (300 plants m-2). Plants were grown under SD and photos were taken 35 days after sowing.
Kaempferol--Rham-Glc
Kaempferol--Rham-Glc-Rham
Kaempferol--Coum-Rham-Rham
Looking for compounds which acount for the most variability
What is it useful for?
- to compare “all” metabolites in various genotypes or treatments
- to find compounds which are affected the most
- to identify and quantify interesting candidates