Low lightHigh 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

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 precursor ion product ions MLKIKRVPTVVSNYQKDDGAEDPVGCGRNCLGACCLNGARLPLYACKNLVKSGEKLVISHEAIEPPVAFL ESLVLGEWEDRFQRGLFRYDVTACETKVIPGKYGFVAQLNEGRHLKKRPTEFRVDKVLQSFDGSKFNFT KVGQEELLFQFEAGEDAQVQFFPCMPIDPENSPSVVAINVSPIEYGHVLLIPRVLDCLPQRIDHKSLLLAV HMAAEAANPYFRLGYNSLGAFATINHLHFQAYYLAMPFPLEKAPTKKITTTVSGVKISELLSYPVRSLLFE GGSSMQELSDTVSDCCVCLQNNNIPFNILISDCGRQIFLMPQCYAEKQALGEVSPEVLETQVNPAVWEIS GHMVLKRKEDYEGASEDNAWRLLAEASLSEERFKEVTALAFEAIGCSNQEEDLEGTIVHQQNSSGNVN QKSNRTHGGPITNGTAAECLVLQ* counts acquisition time (mins)

6x His TRYPSIN detect by mass spectrometry precursor ions product ions * * * * * * * * * * * *** FRAGMENTATION in vitro translation with labelled amino acid (e.g. 13 C-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