title: Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder.
abstract: We analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC-MS/MS strategies, 512 phosphorylation sites on 540 non-redundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, and this enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside of structural protein domains (97%) but were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, we found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs, linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is significantly depleted in protein domains and significantly enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This supports the hypothesis that disordered regions in proteins allow kinases, phosphatases, and phosphorylation-dependent binding proteins to gain access to target sequences to regulate local protein conformation and activity.
authors.affiliation: Proteomic Mass Spectrometry, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB101SA, United Kingdom.
authors.forename: Mark O
authors.initials: MO
authors.lastname: Collins
authors.forename: Lu
authors.initials: L
authors.lastname: Yu
authors.forename: Iain
authors.initials: I
authors.lastname: Campuzano
authors.forename: Seth G N
authors.initials: SG
authors.lastname: Grant
authors.forename: Jyoti S
authors.initials: JS
authors.lastname: Choudhary
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D016000:Cluster Analysis; D003600:Cytosol / Q000737:chemistry*; D051379:Mice; D019636:Neurodegenerative Diseases / Q000209:etiology; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D020539:Sequence Analysis, Protein
chemical_list: D010750:Phosphoproteins; D020543:Proteome