Fumarate Chemoproteomics Database
Documentation
This document is designed to provide a brief introduction to a searchable database of FH-regulated cysteines identified by Kulkarni et al. (“Chemoproteomic portrait of the oncometabolite fumarate,” biorxiv.org, 2018).
Methodology
Proteome-wide cysteine reactivity was profiled by applying iodoacetamide alkyne as previously reported (Weerapana et al., Nature, 2010, Qian et al., Chembiochem, 2013). This method involves the treatment of paired samples (i.e. experimental and control) with iodoacetamide alkyne. Each sample is individually conjugated to isotopically distinguishable azide biotin tags using click chemistry, and then pooled, enriched over streptavidin, and subjected to on-bead tryptic digest. Cysteine-containing peptides labeled by iodoacetamide alkyne are released by cleavage of an azobenzene linker and identified by LC-MS/MS. The relative intensity ratio (ratio, R) of light/heavy isotopic pairs in the MS1 spectra provides a quantitative readout of the extent of Cys-labeling (relative “stoichiometry”) in each sample. “FH-regulated cysteines” are protein cysteine residues whose reactivity is altered in UOK262 (FH-deficient, high fumarate) cells, as compared to UOK262 WT (FH-rescued, lower fumarate) cells.
Input
Each list may be browsed and sorted by field by clicking on the column headers. In addition, users may query specific properties of the database using the following inputs:
- Official Gene Symbol
- Uniprot Accession ID
- L/H Ratio (higher values indicate reduced reactivity upon fumarate-treatment or FH mutation)
Output
The database outputs the following columns:
- ID: Uniprot Accession number
- Gene symbol: Official gene symbol
- Description: full gene name
- Subcellular location: annotated subcellular location
- Sequence: sequence of peptide identified
- Position: position of modified cysteine residue
- Function: annotated function of cysteine residue (if any)
- Run 1 - Run 4: L/H ratios in individual LC-MS/MS runs
- L/H ratio Avg: ratio of light and heavy MS1 ion intensities for iodoacetamide alkyne enrichment. Higher values indicate a cysteine is less reactive after fumarate-treatment (“fumarate-sensitive cysteines”) or FH mutation (FH-regulated cysteines)
- Std. Dev.: standard deviation of L/H ratio for replicate measurements
- N: number of replicates average measurement was based upon