Summary
Adenosine-to-inosine RNA editing is one of the most common types of RNA editing, a posttranscriptional modification made by special enzymes. We present a proteomic study on this phenomenon for Drosophila melanogaster. Tree deep proteome data sets for Canton-S fruit fly line were used in the study: two taken from public repository and the third one obtained here for the isolated brains. A customized protein sequence database was generated using results of genome-wide adenosine-to-inosine RNA studies in fruit fly and applied for identifying the edited proteins. The total number of 56 edited proteins was found in all data sets, 7 of them being shared between the whole insect, head and brain proteomes. Two edited sites in syntaxin 1A (Syx1a) and complexin (cpx) belonging to a presynaptic vesicle fusion complex were selected for validation by targeted analysis. The results obtained for two selected peptides using Multiple Reaction Monitoring have shown remarkably constant ratios of unedited-to-edited protein variants in flies raised under different ambient temperatures of 10, 20 and 30°C. Specifically, these ratios were 34.5:1 and 2.1:1 for Syx1a and cpx, respectively. The work demonstrates the feasibility to identify the RNA editing event at the proteome level using shotgun proteomics and customized edited protein database.
- BCA
- bicinchoninic acid
- TEABC
- triethylammonium bicarbonate
- IAM
- iodoacetamide
- rpm
- rounds per minute
- HCD
- higher-energy collisional dissociation
- VAI
- Variant Annotation Integrator
- RADAR
- rigorously annotated database of A-to-I RNA editing
- ADAR
- adenosine deaminase, RNA specific.
- FDR
- false discovery rate
- PSM
- peptide spectrum match
- Fmoc
- 9-fluorenyl methyloxy carbonyl
- OtBu
- tert-butyl
- DMF
- dimethylformamide
- HCTU
- O-(1H-6-Chloro-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate
- TMP
- 2,4,6-Trimethylpyridine
- TIS
- Triisopropylsilane
- QqQ
- triple quadruplole
- CV
- coefficient of variance
- SNP
- single nucleotide polymorphism
- SNARE
- SNAP (Soluble NSF Attachment Protein) REceptor
- GO
- gene ontology
- MRM
- multiple reaction monitoring
- CE
- collision energy