Download Dealing with heterogeneity

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?
– 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
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
Protein abundance – VTC2
MLKIKRVPTVVSNYQKDDGAEDPVGCGRNCLGACCLNGARLPLYACKNLVKSGEKLVISHEAIEPPVAFL
ESLVLGEWEDRFQRGLFRYDVTACETKVIPGKYGFVAQLNEGRHLKKRPTEFRVDKVLQSFDGSKFNFT
KVGQEELLFQFEAGEDAQVQFFPCMPIDPENSPSVVAINVSPIEYGHVLLIPRVLDCLPQRIDHKSLLLAV
HMAAEAANPYFRLGYNSLGAFATINHLHFQAYYLAMPFPLEKAPTKKITTTVSGVKISELLSYPVRSLLFE
GGSSMQELSDTVSDCCVCLQNNNIPFNILISDCGRQIFLMPQCYAEKQALGEVSPEVLETQVNPAVWEIS
GHMVLKRKEDYEGASEDNAWRLLAEASLSEERFKEVTALAFEAIGCSNQEEDLEGTIVHQQNSSGNVN
QKSNRTHGGPITNGTAAECLVLQ*
counts
precursor ion
product ions
acquisition time (mins)
Protein abundance – VTC2
in vitro translation with labelled
amino acid (e.g. 13C-Leu*)
*
*
*
*
*
Detect mass difference
between labelled and
unlabelled protein
precursor ions
TRYPSIN
*
*
*
*
*
FRAGMENTATION
*
*
*
detect by mass
spectrometry
*
product ions
• 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
Unlabelled glutamate (Glu) and glutamine (Gln)
Abundance
1600
1400
Glu
1200
Gln
1000
800
600
400
200
0
0
20
40
60
80
100
120
140
Tim e after adding 15NH3 (m in)
15N-labelled Glu and Gln
3000
2500
Abundance
2000
15N-Glu
1500
15N-Gln (1)
15N-Glu (2)
1000
500
0
0
20
40
60
80
100
120
-500
m e after adding 15NH3 (m inTim e after adding 15NH3 (m in)
140