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发布日期: Jun 1, 2020
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Integrated omics in Drosophila uncover a circadian kinome

Results .Quantifying multi-omics data under circadian cycles . To investigate global phosphorylation in flies, we conducted quantitative proteomics and phosphoproteomics using the Tandem Mass Tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) on WT and per 0fly heads collected at 3?h intervals on 2 days under constant darkness (DD) condition (Fig.? 1a ). Altogether we identified 61,460 non-phosphorylated peptides and 12,465 phosphopeptides from 32 samples. The majority of the peptides (35,280; 57.40%) and phosphopeptides (8193; 65.73%) could be matched with ≥2 spectral counts, whereas the average spectral counts were 2.5 and 4.4 for all peptides and phosphopeptides, respectively (Fig.? 1b ). We next mapped non-phosphorylated peptides to their corresponding protein sequences, and obtained 5998 and 6034 proteins in WT and per 0flies, respectively (Supplementary Data? 1 ). Only 14.87% (912) of 6134 quantified proteins were assigned with one matched peptide, with an average number of 8.6 quantified peptides per protein (Fig.? 1c ). We also mapped phosphopeptides to full-length protein sequences and in total obtained 3295 phosphoproteins with 14,946 non-redundant p-sites from all 32 samples with an average p-site localization probability of 0.91, including 12,399 p-Ser (82.96%), 2458 p-Thr (16.45%), and 89 p-Tyr (0.60%) sites (Fig.? 1d, e and Supplementary Data? 1 ). We compared the p-sites identified here with eight public databases, including dbPAF11, dbPTM12, Phospho.ELM13, PHOSIDA14, PhosphoPep15, PhosphoSitePlus16, SysPTM17, and UniProt18. Only 37.56% p-sites quantified in this study were annotated and included in at least one phosphorylation database, whereas up to 9333 p-sites have never been reported (Fig.? 1f ). By using two-sided hypergeometric test, the enrichment analysis of Gene Ontology (GO) terms revealed that proteins expressed in the head are mainly involved in neurotransmitter secretion, translation, transport, and splicing, whereas phosphorylation is enriched in pathways that regulate GTPase activity, olfactory learning, chemical synaptic transmission, and intracellular signaling, as well as protein phosphorylation (Supplementary Fig.? 1a ).Fig. 1: Circadian multi-omics profiling of fly heads.a WT and per 0fly heads were collected at 3?h intervals on 2 days in DD. LC-MS/MS-based proteomics and phosphoproteomics as well as RNA-seq-based transcriptomics were conducted. An integrative pipeline iCMod was implemented for analyzing the multi-omics data. NCPs were detected and their corresponding protein kinases were predicted. Locomotor rhythm analysis was employed for validation of the predicted kinases. b The distributions of raw MS/MS spectral counts of peptides and phosphopeptides quantified from proteomics and phosphoproteomics data, respectively. c The distribution of peptide numbers quantified from proteomics data. d The number of phosphoproteins, as well as p-Ser, p-Thr, and p-Tyr residues identified in each sample. e The distribution of the amino-acid residues (left) and the assigned localization probability (right) for all detected p-sites. f Comparison of p-sites detected in this study with known p-sites curated in public databases. g The Spearman’s rank correlations of transcriptomes, proteomes, and phosphoproteomes detected on Day 1 and Day 2, respectively. Full size image To further validate the proteomic and phosphoproteomic data sets, we conducted transcriptome profiling of WT and per 0fly heads collected in DD by RNA sequencing (RNA-seq) (Fig.? 1a ). Over 9.4?×?10 8 reads were sequenced in all 32 samples, with an average of 3.1?×?10 7 and 2.9?×?10 7 reads in WT and per 0, respectively (Supplementary Fig.? 1b ). After reads mapping and transcript assembly, there are 15,280 and 14,760 mappable protein-coding genes identified in WT and per 0flies, respectively, which occupy 70.84% of the fly protein-coding transcriptome (Supplementary Fig.? 1c and Supplementary Data? 1 ). Fragments Per Kilobase of exon per Million fragments mapped (FPKM) values were calculated for the quantification of individual mRNAs, and the average FPKM values are 114 and 118 for WT and per 0flies, respectively (Supplementary Fig.? 1d and Supplementary Data? 2 ). Similar to proteomic data, mRNAs expressed in the head are also enriched in the splicing process, as well as axon guidance, development and transcription (Supplementary Fig.? 1a ).To ensure data quality, only proteins and p-sites quantified in all 16 samples of WT or per 0flies were retained (Supplementary Fig.? 1e–g ). In total, there are 4537 proteins and 5724 p-sites quantified in all WT flies, whereas 4561 proteins and 5739 p-sites were quantified in all per 0samples. The multi-omics measurements show high reproducibility, with Spearman’s rank correlation coefficients of 0.99, 0.95, and 0.85 at mRNA expression, protein expression, and phosphorylation levels for the two cycles monitored, respectively (Fig.? 1g ). After normalization of the proteomic and phosphoproteomic data (Supplementary Fig.? 1h ), we analyzed the correlation among the multi-omics data, and found the correlation of temporal variation between proteome and phosphoproteome is much higher than that between transcriptome and proteome (Supplementary Figs.? 2 , 3 , 4a and Supplementary Note 1). These results suggest that phosphorylation has a major role in regulating the temporal alterations of protein level.Employing iCMod for integrating circadian multi-omics data . In this work, we implemented a pipeline termed iCMod to integrate circadian multi-omics data, and to accurately predict mRNAs, proteins and p-sites with circadian oscillation (Fig.? 2 ). For a protein detected by proteomics/phosphoproteomics analysis, we believe its corresponding mRNA should be readily detectable in the transcriptomic data. For 16.70% and 16.80% of quantified proteins from WT and per 0flies, respectively, we observed that their corresponding mRNAs are expressed at a low level with FPKM?