While the proteratos created from S also ft to a Gaussadstrbutobut wth a lower R worth and the dstrbutoof the ratos s very much broader ndcatng extra varatothe values, whch ndcates that the quantfcatos much less precise.dentfcatoof phosphoproteome and bonformatc analyss of phosphorylatostes We enrched phosphopeptdes usng a combnedhC MAC approach19 from total rat cortcal neuronal cell lysates immediately after mxng wth the SAM rat brahomogenates.We used a 10 ppm precursor omass tolerance for all phosphorylated peptdes as well as a false dscovery fee with the peptde degree decrease tha1%.From two techncal replcates of the phosphopeptde enrched neuronal lysates, we dentfed betwee1525 and 2936 14unque phosphopeptdes wth a phosphopeptde enrchment rato betwee51% and 69%.The number of correspondng unque phosphoprotens dentfed ranged from 647 to 846.Searchng by 15the same MudPT analyss, we dentfed 331 to 728 phosphopeptdes correspondng to 178 to 344 15phosphoprotens.The phosphopeptde enrchment ratos also ranged betwee49% and 65%.
The smar enrchment ratos kinase inhibitor SAR245409 betweethe cultured neurons along with the bratssue propose that ourhC MAC technique to enrch phosphopeptdes s not based mostly towards etherhomogeneous neuronal cells orhghly complex bratssue.ether the neurons or the bratssue, the phosphorylatoevents on the three resdues are 2 3% at tyrosne, 15 17% at threonne, and 81 82% at serne.Ths dstrbutos consstent wth all prevous huge scale studes to dentfy phosphorylatostes18, 35.We carried out Alisertib a replcate bologcal experment usng the samehC MAC process to enrch for phosphopeptdes and pulled all phosphopeptde dentfcatons nto 3 groups, handle neurons, PCneurons, and also the bratssue.The total numbers of phosphopeptde dentfcatons these groups are 15247, 14139, and 6948, respectvely,and also the total numbers of phosphorylatostes are 18239, 16824, and 7785, respectvely.Usng a phosphorylatoste localzatoalgorthm based mostly obnomal probabty29, we assgned a localzatoprobabty score for every ste, and found that we could obtastatstcally sgnfcant scores for two three of the stes.
After

removng redundancy we confdently dentfed 3370, 2762 and 1408 unque phosphorylatostes from the 3 respectve groups wth the unototalng 4647 stes.All phosphopeptdes and ther ste localzatoresults are lsted Table S7.To estmate the abundance of phosphorylatostes wtheach sample we implemented spectral countng to represent the amount of tmes a peptde s analyzed by the mass spectrometer.Spectral counts correlate wth proteabundance36, andhave also beeappled to approxmate the abundance of phosphorylatostes mouse tssues37.The 4647 stes that were confdently localzed from all three sample categores were analyzed byherarchcal clusterng wth spectral countng as the cluster metrc, and presented as aheat maFgure 3D and Table S1.Not surprsngly, the dstrbutopatterof phosphorylatoste abundance betweecontrol neurons and PCtreated neurons shows far even more smarty thabetweeneurons and the bran, ndcatng that the brahas dstnct phosphorylatopatterns compared to dssocated neurons.