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)