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High light response in Arabidopsis thaliana. 4 days. 1100 transcripts change. Low light. High light. Anthocyanin light response mutant. Mutant characterisation by metabolite profiling. Targeted analysis of anthocyanins and other flavonoids (MRM). What else is different? - PowerPoint PPT Presentation
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Low light High light
High light response in Arabidopsis thaliana
4 days
1100 transcripts change
Anthocyanin light response mutant
Mutant characterisation by metabolite profiling
• Targeted analysis of anthocyanins and other flavonoids (MRM).
• What else is different?– Run samples (wild type v. mutant HL v. LL) on LC-QToF.– Identify potential compounds in each sample using molecular
feature extraction (MFE) in MassHunter: ~3000 “compounds”.– Align features (mass/retention time pairs) across samples
(Perera-Yang method).– Statistical analysis to identify differentially expressed features.
• Check MFE data to confirm.• Identify features from accurate mass/isotope abundance and
MS/MS spectra.• Use m/z, retention time and MS/MS data to set up specific assays
by MRM using QQQ.
Total ion chromatograms of 4 samples
Extracted ion chromatograms (EIC) from one sample
z
1
1
1
1
345.0457
90.2
53.1
80.4
66.6
97.4
514.1
51.8
180.2
241.4
70
1846.7
13727.8
440.7
130.0635
185.1594
191.5252 62.6
195.5933
325.0105
344.0428
199.0172
328.0761
130.0648
38.9636
73.0804
86.0952
MS/MS Spectrum Peak Listm/ z
328.0785
130.0646
130.0619
164.0706
Abund
32270.4
11430.4
84.3
Molecular feature extraction- identifies “compounds” by amalgamating charge states and adducts with the same chromatographic retention time.
Extracted compounds aligned across samples and compared.
Hierarchical clustering compares abundance of compounds in mutant and WT plants grown in low and high light
Extracted compounds aligned across samples and compared.
Hierarchical clustering compares abundance of compounds in mutant and WT plants grown in low and high light
RT7.687
AlgorithmFind by Molecular Feature
Mass610.1536
Compound LabelCpd 19: 7.687
m/z611.1607
Protein abundance – VTC2
precursor ion
product ions
MLKIKRVPTVVSNYQKDDGAEDPVGCGRNCLGACCLNGARLPLYACKNLVKSGEKLVISHEAIEPPVAFLESLVLGEWEDRFQRGLFRYDVTACETKVIPGKYGFVAQLNEGRHLKKRPTEFRVDKVLQSFDGSKFNFTKVGQEELLFQFEAGEDAQVQFFPCMPIDPENSPSVVAINVSPIEYGHVLLIPRVLDCLPQRIDHKSLLLAVHMAAEAANPYFRLGYNSLGAFATINHLHFQAYYLAMPFPLEKAPTKKITTTVSGVKISELLSYPVRSLLFEGGSSMQELSDTVSDCCVCLQNNNIPFNILISDCGRQIFLMPQCYAEKQALGEVSPEVLETQVNPAVWEISGHMVLKRKEDYEGASEDNAWRLLAEASLSEERFKEVTALAFEAIGCSNQEEDLEGTIVHQQNSSGNVNQKSNRTHGGPITNGTAAECLVLQ*
cou
nts
acquisition time (mins)
6x H
is TRYPSIN
detect by mass spectrometry
precursor ions
product ions
**
*
**
** *
**
* * * *
FRAGMENTATION
in vitro translation with labelled amino acid (e.g. 13C-Leu*)
• Spike plant extracts with a known quantity of labelled VTC2
• Directly compare abundance with VTC2 present in leaf extract
• Spike plant extracts with multiple biosynthetic proteins – simultaneous quantification
Detect mass difference between labelled and unlabelled protein
Protein abundance – VTC2
Unlabelled glutamate (Glu) and glutamine (Gln)
0
200
400
600
800
1000
1200
1400
1600
0 20 40 60 80 100 120 140
Tim e after adding 15NH3 (m in)
Ab
un
dan
ce
Glu
Gln
15N-labelled Glu and Gln
-500
0
500
1000
1500
2000
2500
3000
0 20 40 60 80 100 120 140
m e after adding 15NH3 (m inTim e after adding 15NH3 (m in)
Ab
un
dan
ce 15N-Glu
15N-Gln (1)
15N-Glu (2)