Label-free proteomic comparison reveals ciliary and non-ciliary phenotypes of IFT-A mutants.
Janelle C LeggereJaime V K HibbardOphelia PapoulasChanjae LeeChad G PearsonEdward M MarcotteJohn B WallingfordPublished in: bioRxiv : the preprint server for biology (2023)
DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila . A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.
Keyphrases
- label free
- mass spectrometry
- high throughput
- high resolution
- protein protein
- amino acid
- binding protein
- liquid chromatography
- induced apoptosis
- transcription factor
- genome wide
- single cell
- oxidative stress
- cell death
- ms ms
- cell proliferation
- endoplasmic reticulum stress
- gas chromatography
- cell free
- capillary electrophoresis
- tandem mass spectrometry
- wild type
- quantum dots
- fluorescence imaging