Integrated proteomic analysis of post-translational modifications by serial enrichment

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1 Supplementary Information: Integrated proteomic analysis of post-translational modifications by serial enrichment Philipp Mertins; Jana W Qiao; Jinal Patel; Namrata D Udeshi; Karl R Clauser; DR Mani; Michael W Burgess; Michael Gillette; Jacob D Jaffe; Steven A Carr The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA Supplementary Item Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Table 1 Supplementary Table 2 Supplementary Table 3 Supplementary Table 4 Supplementary Table 5 Supplementary Table 6 Supplementary Table 7 Title Experimental workflow for proteome and SEPTM analysis Uniqueness per fraction and occurrence of modified peptides per high ph RP fraction Overlap of proteins/sites in replicate experiments The proteasome inhibitor Velcade strongly induces protein ubiquitination, upregulates specific proteins and phosphosites, but does not affect lysine-acetylation List of all quantified proteins, psty-, K(GG)-, and K(Ac)- sites that were quantified in at least 2 replicates in the high, medium and low coverage experiments List of all quantified peptides observed in the proteome analysis at high, medium and low coverage of one representative replicate (replicate 1) List of all quantified peptides observed in the phosphoproteome, ubiquitinome and acetylome analyses at high, medium and low coverage of one representative replicate (replicate 1) Ubiquitin- and acetyl-site tables for label-free and SILACbased comparison of non-serial and serial enrichments Frequency of post-translationally modified peptides in whole proteome datasets and occurrence of peptides with multiple different PTMs in PTM-enriched datasets Gene-enrichment analysis of proteins regulated on ubiquitin or phosphorylation level Kinases with regulated phosphorylation-sites after Velcade treatment

2 Supplementary Figure 1: A light heavy 24 fractions 5% 24 proteome fractions LC-MS/MS DMSO Velcade 95% Mix protein lysates Reduction/ Alkylation tryptic digest basic RP separation combine into 12 fractions 12 phosphoproteome IMAC enrichments PO 4- - Fe 3+ IMAC flow-through combine into 6 fractions 6 ubiquitinome K(GG) enrichments GG-K Y flow-through 6 acetylome K(Ac) enrichments SEPTM - Serial Enrichment of PTMs Ac-K Y LC-MS/MS B High ph reversed phase separaon [ph 10.0] 72 total fracons fxna combine 1, 25, 49 early, middle & late fracons into 24 fracons 24, 48, 72 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Supplementary Figure 1: Experimental workflow for proteome and SEPTM analysis (a) Jurkat cells were SILAC labeled for relative quantification and treated with the proteasome inhibitor Velcade at 1 µm for 4h. After tryptic digest peptides were separated by basic reversed-phase (RP) chromatography and sequentially enriched for phosphorylated peptides with IMAC, for ubiquitinated peptides with K(GG)-specific antibodies and for lysine-acetylated peptides with K(Ac)-specific antibodies. (b) Basic RP fractions were combined in a non-contiguous way to cover a wide range of hydrophilic to hydrophobic peptides in each concatenated fraction. Early eluting hydrophilic peptides were collected in fraction A.

3 Supplementary Figure 2: Phosphoproteome 2.39% 0.78% 15.11% 80.83% 1 fxn 2 fxns 3 fxns 4 fxns Ubiquinome Acetylome 8.73% 0.82% 0.27% 8.62% 0.84% 0.32% 89.88% 1 fxn 2 fxns 3 fxns 4 fxns 90.02% 1 fxn 2 fxns 3 fxns 4 fxns Supplementary Figure 2: Uniqueness per fraction and occurrence of modified peptides per high ph RP fraction Pie charts representing the percentage of distinct peptides identified in one, two, three or four psty-, K(GG)-, and K(Ac)-fractions. Percentage values shown are averages of the three high-scale study replicates. Phosphoproteome, ubiquitinome, and acetylome fractions were acquired as described in the Online Methods section. High percentage of distinct peptides found in only one PTM fraction is indicative of unique peptide landscape in each fraction.

4 Supplementary Figure 3: Proteins Rep1 (8,038) Rep2 (7,860) Rep3 (7,792) K(GG)-sites Rep1 (12,803) Rep2 (17,664) Rep3 (15,757) psty-sites Rep1 (21,857) Rep2 (22,383) Rep3 (18,161) K(ac)-sites Rep1 (2,982) Rep2 (3,638) Rep3 (2,949) Supplementary Figure 3: Overlap of proteins/sites in replicate experiments Our phosphoproteome analysis yielded 20,800 quantified and fully localized phosphorylation sites per replicate, 13,701 of which (representing 4,486 phosphoproteins) were detected and quantified in all three replicates. The ubiquitome analysis revealed 15,408 localized K(GG)-sites per replicate with an overlap of 44% in the 3 replicates. The overlapping 6,759 K(GG) sites were measured on 2,631 proteins. The acetylation analysis resulted in the smallest number of sites with 3,190 per replicate and 1,757 in all three replicates on 971 proteins. Note that all PTM-sites were fully localized in each experiment.

5 Supplementary Figure 4: A X (Rep1) Y (Rep2) X (Rep1) Y (Rep3) X (Rep2) Y (Rep3) B Transcription initiation Proteasome Proteome psty K(GG) K(Ac) Log 2 SILAC ratios Velcade treated/untreated POLR2B TAF2G HCFC1 GTF2E2 POLR2A POLR2E POLR2C TBP IKBKAP GTF2H3 ERCC3 SKP2 CDK4 ACTL6A MYC RB1 CCND3 Cell Cycle Replication RPA1 MCM10 MCM7 MCM4 MCM3 MCM5 PCNA RFC3 SAP30 YY1 SP1 YWHAQ CTNNB1 CDK1 PPP2R1A RFC5 NCOR1 RBBP7 RFC2 PRIM1 PRIM2 RPA3 POLA2 Protein K(GG) MTA2 GATAD2B HDAC1 Transcription MTOR PML CDC25B XRCC6 HYRC XRCC5 USF1 BUB3 PSMA1 PSMB10 PSMB5 PSMA3 PSMA4 PSMB1 PSMA7PSMD8 PSMD14 PSMC3 PSMA5 PSMC2 RPL35 RPL27A RPL9 RPS17 RPL4 RPL5 RPS19 RPS9 RPS5 RPLP0 RPS10 EIF5 RPS3 EEF2 EIF3B EIF3D USP7 NUP98 USP11 CS PSMB3 PSMA2 PSMD4 USP14 RPL3 RPL34 USP3 CCT3 CCT8 EIF3H Ribosome psty TCP1 CCT5 psty & K(GG) Supplementary Figure 4: The proteasome inhibitor Velcade strongly induces protein ubiquitination, upregulates specific proteins and phosphosites, but does not affect lysine-acetylation (a) Scatter plots show protein and PTM-site Log SILAC ratios (Velcade treated/untreated) that were measured in all 3 replicates (X, Y indicate x- and y-axis; Rep1//3 = replicates 1, and 3; psty = phosphorylated peptides; K(GG) = ubiquitinated peptides; K(Ac) = acetylated peptides). Only fully localized PTM-sites that were normalized to their corresponding protein ratios are plotted. Significantly regulated proteins/ptm-sites with a moderated T-test p-value <0.0 are shown in red. (b) Co-regulation of cellular processes by Velcade-induced changes in protein abundance, ubiquitination and phosphorylation. All proteins that were significantly upregulated on protein or PTM-site level were subjected to network analysis using the STRING database 18 and all higher order protein nodes with 3 or more connections that were derived from curated databases were visualized using Cytoscape.

6 Supplementary Table 5: Number of quaned disnct pepdes overlapping in 3 high coverage experiments: psty 1 K(GG) 2 K(Ac) 3 all pepdes Proteome dataset psty psty&k(gg)* psty&k(ac)* psty&k(gg)&k(ac)* Phosphoproteome dataset K(GG) K(GG)&pSTY* K(GG)&K(Ac)* K(GG)&pSTY&K(Ac)* Ubiquinome dataset K(Ac) K(Ac)&pSTY* K(Ac)&K(GG)* K(Ac)&pSTY&K(GG)* Acetylome dataset ) psty: pepdes phosphorylated either on serine, threonine or tyrosine residues 2) K(GG): pepdes modied with a glycine-glycine stub on lysine residues 3) K(Ac): pepdes modied with acetyl on lysine residues * Pepdes with mulple dierent post-translaonal modicaons on the same pepde Supplementary Table 5: Frequency of post-translationally modified peptides in whole proteome datasets and occurrence of peptides with multiple different PTMs in PTM-enriched datasets High coverage proteome and PTM datasets were searched simultaneously for psty, K(GG) and K(Ac) modifications. Only distinct peptides that were quantified in all 3 replicates were counted.

7 Supplementary Table 6: Up-regulated K(GG)-proteins: GO-term Count cell cycle 163 RNA biosynthetic process 53 translational elongation 60 protein complex assembly 89 DNA metabolic process 108 transcription 51 glucose metabolic process 25 fatty acid metabolic process 21 Up-regulated psty-proteins: GO-Term Count spindle organization 8 regulation of cell cycle 10 transcription 64 nuclear transport 12 cytoskeleton organization 19 Down-regulated K(GG)-proteins: GO-term Count chromatin assembly 11 cell motion 17 translation 29 negative regulation of apoptosis 15 cell morphogenesis 12 Gene-enrichment analysis (p<0.05) Supplementary Table 6: Gene-enrichment analysis of proteins regulated on ubiquitin or phosphorylation level Gene-enrichment analysis was performed using DAVID Bioinformatic Resource (6.7). Significantly regulated K(GG) and psty-sites (moderated T-test pval<0.02) were analyzed with the corresponding full PTM dataset as a background list. Categories with a modified Fisher Exact p-value <0.05 are shown.

8 Supplementary Table 7: Uniprot# Protein name Gene name site Pepde sequence site rao 1 modt pval 2 protein rao 3 E7EUM9 Cyclin-dependent kinase 12 CDK12 S614 TQVS(ph)VTAAIPHLK E7EUM9 Cyclin-dependent kinase 12 CDK12 S334 SSS(ph)PFLSK E7EUM9 Cyclin-dependent kinase 12 CDK12 S681 HLLTDLPLPPELPGGDLS(ph)PPDS(ph)PEPK Q13177 Serine/threonine-protein kinase PAK 2 PAK2 S58 IISIFS(ph)GTEK Q8TDX7 Serine/threonine-protein kinase Nek7 NEK7 S204 TTAAHSLVGTPYYMS(ph)PER P49137 MAP kinase-acvated protein kinase 2 MAPKAPK2 T222 ETTSHNSLT(ph)T(ph)PCYTPYYVAPEVLGPEK P49137 MAP kinase-acvated protein kinase 2 MAPKAPK2 T334 VPQT(ph)PLHTSR Q16644 MAP kinase-acvated protein kinase 3 MAPKAPK3 T201 ETTQNALQT(ph)PCYTPYYVAPEVLGPEKYDK F5H2M7 Serine/threonine-protein kinase WNK1 WNK1 S2619 VPPAVIIPPAAPLS(ph)GRR F5H2M7 Serine/threonine-protein kinase WNK1 WNK1 S1759 FIVS(ph)PVPESR P78527 DNA-dependent protein kinase catalyc subunit PRKDC S2612 STVLT(ph)PMFVETQAS(ph)QGTLQTR P43403 Tyrosine-protein kinase ZAP-70 ZAP70 T293 IDTLNSDGYT(ph)PEPAR Q8TD19 Serine/threonine-protein kinase Nek9 NEK9 S793 GMEGLIS(ph)PTEAMGNSNGASSSCPGWLR Q8TD19 Serine/threonine-protein kinase Nek9 NEK9 T886 GTPLT(ph)PPACACSSLQVEVER Q8TD19 Serine/threonine-protein kinase Nek9 NEK9 T333 SST(ph)VTEAPIAVVTSR E9PC69 Serine/threonine-protein kinase MARK2 MARK2 S390 PSADLTNSSAPS(ph)PSHK Q14680 Maternal embryonic leucine zipper kinase MELK T415 SLT(ph)PALCRT(ph)PANK Q14680 Maternal embryonic leucine zipper kinase MELK S498 LMTGVIS(ph)PERR P33981 Dual specicity protein kinase TTK TTK S281 VPVNLLNS(ph)PDCDVKTDDSVVPCFMK P33981 Dual specicity protein kinase TTK TTK S821 YVLGQLVGLNS(ph)PNSILK P33981 Dual specicity protein kinase TTK TTK T33 FKNEDLT(ph)DELSLNK P33981 Dual specicity protein kinase TTK TTK S393 RKS(ph)ECINQNPAASSNHWQIPELAR O95747 Serine/threonine-protein kinase OSR1 OXSR1 S359 AAISQLRS(ph)PR P36507 Dual specicity mitogen-acvated protein kinase kinase 2 MAP2K2 S23 RKPVLPALTINPTIAEGPS(ph)PTSEGASEANLVDLQK Q13546 Receptor-interacng serine/threonine-protein kinase 1 RIPK1 S320 RMQS(ph)LQLDCVAVPSSR P46734 Dual specicity mitogen-acvated protein kinase kinase 3 MAP2K3 S3 (ac)mes(ph)passqpasmpqsk P49674 Casein kinase I isoform epsilon CSNK1E S408 IPASQTS(ph)VPFDHLGK Q9BUB5 MAP kinase-interacng serine/threonine-protein kinase 1 MKNK1 T385 GLPT(ph)PQVLQR P51957 Serine/threonine-protein kinase Nek4 NEK4 S484 LLGSSDS(ph)PASASR P12931 Proto-oncogene tyrosine-protein kinase Src SRC S75 LFGGFNSSDTVTS(ph)PQR Q9H0K1 Serine/threonine-protein kinase SIK2 SIK2 S379 AQTVGLPVTMHS(ph)PNMR Q9H0K1 Serine/threonine-protein kinase SIK2 SIK2 S576 EVHNRS(ph)PVSFR Q9NRH2 SNF-related serine/threonine-protein kinase SNRK S639 S(ph)AGELVESLK O43683 Mitoc checkpoint serine/threonine-protein kinase BUB1 BUB1 S459 VQPS(ph)PTVHTK O43683 Mitoc checkpoint serine/threonine-protein kinase BUB1 BUB1 S375 EAPPVVPPLANAISAALVS(ph)PATSQSIAPPVPLK Q9NYL2 Mitogen-acvated protein kinase kinase kinase MLT MLTK S302 DLS(ph)FKEQELKER Q2M2I8 AP2-associated protein kinase 1 AAK1 T389 AGQTQPNPGILPIQPALT(ph)PR E7EN19 Mitogen-acvated protein kinase kinase kinase kinase 4 MAP4K4 T909 TMIVHDDVESEPAMT(ph)PSKEGTLIVR E7EN19 Mitogen-acvated protein kinase kinase kinase kinase 4 MAP4K4 S949 LLQIS(ph)PSSGTTVTSVVGFSCDGMRPEAIR H0Y4E8 Serine/threonine-protein kinase SIK3 SIK3 S1020 HS(ph)LTGHSDIR H0Y4E8 Serine/threonine-protein kinase SIK3 SIK3 S1016 HQQVPHILQGLLS(ph)PR Q9H792 Pseudopodium-enriched atypical kinase 1 PEAK1 S861 LVTS(ph)PQSEPPAPFPPPR Q9H792 Pseudopodium-enriched atypical kinase 1 PEAK1 S898 HFTNWTKPTS(ph)PTR Q9H792 Pseudopodium-enriched atypical kinase 1 PEAK1 S587 TIPVKS(ph)PNLSEIK Q16512 Serine/threonine-protein kinase N1 PKN1 S118 RLDLLHQQLQELHAHVVLPDPAATHDGPQS(ph)PGAGGPTCSATNLSR P30291 Wee1-like protein kinase WEE1 T187 LFDT(ph)PHT(ph)PK P42684 Abelson tyrosine-protein kinase 2 ABL2 S915 LGMAGVPEDGEQPGWPS(ph)PAK Q13233 Mitogen-acvated protein kinase kinase kinase 1 MAP3K1 S507 SHDFYSHELSS(ph)PVDSPSSLR E9PC69 Serine/threonine-protein kinase MARK2 MARK2 S570 VPVAS(ph)PSAHNISSSGGAPDR P10398 Serine/threonine-protein kinase A-Raf ARAF S186 QHEAPSNRPLNELLTPQGPS(ph)PR P27448 MAP/microtubule anity-regulang kinase 3 MARK3 S419 RYS(ph)DHAGPAIPSVVAYPK Q86YV5 Tyrosine-protein kinase SgK223 SGK223 S743 VSQGSAESLS(ph)PSFR P53350 Serine/threonine-protein kinase PLK1 PLK1 T210 KKT(ph)LCGTPNYIAPEVLSK O60566 Mitoc checkpoint serine/threonine-protein kinase BUB1 beta BUB1B S684 KLS(ph)PIIEDSR Q96Q15 Serine/threonine-protein kinase SMG1 SMG1 T3573 NLATSADT(ph)PPSTVPGTGK E7ER94 Serine/threonine-protein kinase D2 PRKD2 S375 AQSS(ph)LGYIPLMR Q9UEW8 STE20/SPS1-related proline-alanine-rich protein kinase STK39 S371 RVPGS(ph)S(ph)GHLHK P31749 RAC-alpha serine/threonine-protein kinase AKT1 S129 SGS(ph)PS(ph)DNS(ph)GAEEMEVSLAKPK O60307 Microtubule-associated serine/threonine-protein kinase 3 MAST3 S1102 S(ph)FSSGLHHSLSS(ph)SESLPGSPTHSLSPSPTTPCR ) Average Log2 Velcade/ctrl phosphosite raos across 3 replicates that were normalized by protein expression levels 2) BH corrected moderated T test p-values for phosphosites 3) Average Log2 Velcade/ctrl protein raos across 3 replicates Supplementary Table 7: Kinases with regulated phosphorylation-sites after Velcade treatment. All kinases with regulated phosphorylation sites up to a moderated T p<0.05 are listed.

PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland

PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland AD Award Number: W81XWH-12-1-0248 TITLE: Targeted Inhibition of Tyrosine Kinase-Mediated Epigenetic Alterations to Prevent Resurgence of Castration-Resistant Prostate Cancer PRINCIPAL INVESTIGATOR: Dr.