Dealing with heterogeneity

1
High light response in Arabidopsis thaliana
4 days
1100 transcripts change
Low light
High light
Anthocyanin light response mutant
2
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.

3
Total ion chromatograms of 4 samples
Extracted ion chromatograms (EIC) from one sample
4
Molecular feature extraction- identifies
compounds by amalgamating charge states and
adducts with the same chromatographic retention
time.
5
Extracted compounds aligned across samples and
compared. Hierarchical clustering compares
abundance of compounds in mutant and WT plants
grown in low and high light
6
Extracted compounds aligned across samples and
compared. Hierarchical clustering compares
abundance of compounds in mutant and WT plants
grown in low and high light
7
(No Transcript)
8
(No Transcript)
9
(No Transcript)
10
Protein abundance VTC2
MLKIKRVPTVVSNYQKDDGAEDPVGCGRNCLGACCLNGARLPLYACKNLV
KSGEKLVISHEAIEPPVAFLESLVLGEWEDRFQRGLFRYDVTACETKVIP
GKYGFVAQLNEGRHLKKRPTEFRVDKVLQSFDGSKFNFTKVGQEELLFQF
EAGEDAQVQFFPCMPIDPENSPSVVAINVSPIEYGHVLLIPRVLDCLPQR
IDHKSLLLAVHMAAEAANPYFRLGYNSLGAFATINHLHFQAYYLAMPFPL
EKAPTKKITTTVSGVKISELLSYPVRSLLFEGGSSMQELSDTVSDCCVCL
QNNNIPFNILISDCGRQIFLMPQCYAEKQALGEVSPEVLETQVNPAVWEI
SGHMVLKRKEDYEGASEDNAWRLLAEASLSEERFKEVTALAFEAIGCSNQ
EEDLEGTIVHQQNSSGNVNQKSNRTHGGPITNGTAAECLVLQ
precursor ion
counts
product ions
acquisition time (mins)
11
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





detect by mass spectrometry

FRAGMENTATION




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

12
(No Transcript)