The formation of condensates in membraneless organelles is thought to be driven by protein phase separation. Arginine methylation and serine/threonine phosphorylation are important in the phase separation process; however, these post-translational modifications are often present in intrinsically disordered regions that are difficult to analyze with standard proteomic techniques. To understand their presence and co-occurrence in condensate-associated proteins, here, we use a multiprotease and multi-tandem mass spectrometry (MS/MS) fragmentation approach, coupled with heavy methyl stable isotope labeling of amino acids in cell culture (SILAC) and phospho- or methyl-peptide enrichment. For Saccharomyces cerevisiae, we report a 50% increase in the known arginine methylproteome, involving 15 proteins that are all condensate-associated. Importantly, some of these proteins have arginine methylation on all predicted sites-providing evidence that this modification can be pervasive. We explored whether arginine-methylated, condensate-associated proteins are also phosphorylated and found 12 such proteins to carry phosphorylated serine or threonine. In Npl3, Ded1, and Sbp1, single peptides were found to carry both modifications, indicating a co-occurrence in close proximity and on the same protein molecule. These co-modifications occur in regions of disorder, whereas arginine methylation is typically on regions of disorder that are also basic. For phosphorylation, its association with charged regions of condensate-associated proteins was less consistent, although some regions with multisite phosphorylation sites were strongly acidic. We conclude that arginine-methylated proteins associated with condensates are typically also modified with protein phosphorylation.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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Evidence ID | Analyze ID | File | Description |
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