Nejvíce citovaný článek - PubMed ID 12376573
To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.
- Klíčová slova
- DeltaSILAC, data-independent acquisition, mass spectrometry, phosphomodiform, phosphorylation, protein lifetime, protein turnover, proteomics, pulse SILAC,
- MeSH
- buněčný cyklus fyziologie MeSH
- cyklin-dependentní kinasy genetika metabolismus MeSH
- fosfoproteiny chemie metabolismus MeSH
- fosforylace MeSH
- glutamáty metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- izotopové značení metody MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- peptidy metabolismus MeSH
- peroxiredoxin VI chemie metabolismus MeSH
- proteolýza * MeSH
- proteom genetika metabolismus MeSH
- proteomika metody MeSH
- sekvence aminokyselin MeSH
- sestřihové faktory chemie metabolismus MeSH
- signální transdukce genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- cyklin-dependentní kinasy MeSH
- fosfoproteiny MeSH
- glutamáty MeSH
- peptidy MeSH
- peroxiredoxin VI MeSH
- PRDX6 protein, human MeSH Prohlížeč
- proteom MeSH
- sestřihové faktory MeSH
- SF3B1 protein, human MeSH Prohlížeč
Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post-translational turnover, we devised a strategy combining pulse stable isotope-labeled amino acids in cells (pSILAC), data-independent acquisition mass spectrometry (DIA-MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome-wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.
- Klíčová slova
- DIA mass spectrometry, alternative splicing, protein turnover, proteomics, pulsed SILAC,
- MeSH
- alternativní sestřih MeSH
- HeLa buňky MeSH
- hmotnostní spektrometrie MeSH
- izoformy RNA genetika metabolismus MeSH
- izotopové značení metody MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- protein - isoformy analýza metabolismus MeSH
- proteiny analýza metabolismus MeSH
- proteolýza MeSH
- proteomika metody MeSH
- průběh práce MeSH
- regulace genové exprese u nádorů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- izoformy RNA MeSH
- messenger RNA MeSH
- protein - isoformy MeSH
- proteiny MeSH