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Wong YK, Wang J, Lim TK, Lin Q, Yap CT, Shen HM. O-GlcNAcylation promotes fatty acid synthase activity under nutritional stress as a pro-survival mechanism in cancer cells. Proteomics 2022 35083852
Abstract:
Protein O-GlcNAcylation is a specific form of protein glycosylation that targets a wide range of proteins with important functions. O-GlcNAcylation is known to be deregulated in cancer and has been linked to multiple aspects of cancer pathology. Despite its ubiquity and importance, the current understanding of the role of O-GlcNAcylation in the stress response remains limited. In this study, we performed a quantitative chemical proteomics-based open study of the O-GlcNAcome in HeLa cells, and identified 163 differentially-glycosylated proteins under starvation, involving multiple metabolic pathways. Among them, fatty acid metabolism was found to be targeted and subsequent analysis confirmed that fatty acid synthase (FASN) is O-GlcNAcylated. O-GlcNAcylation led to enhanced de novo fatty acid synthesis activity, and fatty acids contributed to the cytoprotective effects of O-GlcNAcylation under starvation. Moreover, dual inhibition of O-GlcNAcylation and FASN displayed a strong synergistic effect in vitro in inducing cell death in cancer cells. Together, the results from this study provide novel insights into the role of O-GlcNAcylation in the nutritional stress response and suggest the potential of combining inhibition of O-GlcNAcylation and fatty acid synthesis in cancer therapy. This article is protected by copyright. All rights reserved.
O-GlcNAc proteins:
RUXGL, ADAS, DX39A, MYO1C, IPO5, PESC, NOP56, DDX3X, SCD, MGST3, HNRDL, XPO1, SURF4, OGT1, PPM1G, MOT4, DHX15, CYB5B, SERA, HNRPR, BUB3, ACTN4, MYO1B, GANP, HNRPQ, NDUS7, MPU1, H2AY, FLNB, SC22B, SF3B1, U520, UTP20, NU155, ATP5H, RL1D1, MTA2, RTN3, VAPB, IPO7, ACSL3, BAG2, TOM40, LDHA, DHE3, AATM, PGK1, ASSY, LMNA, TFR1, ALDOA, K2C1, G3P, HSPB1, RPN1, AT1A1, ADT2, PCCA, RLA1, RLA0, LA, K1C18, K2C8, ATPB, ENOA, NPM, TPM3, LDHB, PDIA1, ANXA2, TBB5, TRY1, PROF1, SYEP, HS90A, HNRPC, DAF, 4F2, HS90B, ODPA, RU17, VIME, RS17, K2C7, GNAI3, RSSA, LEG1, ROA1, PARP1, PRS56, HS71B, ODP2, THIO, MGST1, CH60, BIP, HSP7C, GTR1, TOP2A, PYC, PABP1, PCNA, ADT3, IMDH2, KCRU, XRCC6, XRCC5, EF2, K1C10, K2C5, PDIA4, PLST, ETFA, MIF, KPYM, ENPL, HNRPL, PLAK, EZRI, NDKA, RS2, DESP, H13, NCPR, AT2A2, DDX5, TCPA, PTN1, ARF4, RL7, RL17, NUCL, GSTM3, FLNA, FBRL, PUR6, UBA1, ROA2, QCR2, SFPQ, PPIB, RS3, SAHH, COF1, MCM3, RS12, ATPA, U2AF2, RL13, S10A4, PTBP1, SYVC, EF1G, STOM, RL10, APEX1, PYR1, CALX, TKT, ERP29, PRDX6, PRDX5, PRDX3, RL12, PDIA3, CPSM, HNRH1, STIP1, L1CAM, PRDX2, P5CR1, DUT, MCM7, GLYM, HSP74, PHB1, RL22, MYH9, SOAT1, DEK, K22E, RL4, LONM, NUP62, GRP75, IF4A3, RL3, RL13A, ARL1, STAT3, MDHM, RFC3, ECHA, SYIC, LAP2A, LPPRC, MATR3, MSH2, GPDM, VDAC2, KI67, BAG6, RL27A, RL5, RS9, STT3A, CAPZB, SYQ, RL29, AT5G3, TCPE, RL34, FAS, TCPG, EFTU, ACADV, TMEDA, NU153, RBP2, CPT1A, SERPH, RL14, TCPQ, TCPD, FXR1, RAB5C, RAB7A, HCFC1, ROA3, 6PGD, HNRPM, IMA1, HNRPF, MSH6, TXTP, ACLY, COPA, MOT1, SYRC, KAD2, P5CS, XPO2, TERA, NP1L1, DSRAD, ATPK, TMM33, TPIS, MYL6, IF4A1, RS20, S10AA, RAP1B, RL15, RL37A, HNRPK, RS8, RS16, 1433E, RS14, RS23, RS11, RUXE, RL7A, RS4X, RS6, H4, RAB1A, RAN, RL23, RS25, RS26, RL10A, RL11, RL8, PPIA, RS27A, RSMN, RACK1, ACTG, UBC9, TBA1B, TBB4B, GTF2I, TCPB, PRKDC, RL24, ARF5, RL19, SRSF3, MPCP, CLH1, HNRPU, SPTB2, EXOSX, RL18A, RL6, IF4G1, K1C17, PRDX1, RL18, C1QBP, KHDR1, DHX9, NCBP1, AHNK, NU160, SF3A3, ILF3, ACACA, PRDX4, CBX3, TIF1B, SPTN1, HNRPD, SAFB2, TTL12, CAPR1, ITPR1, RRP1B, GANAB, LBR, GOGB1, IMB1, NUMA1, SUZ12, U5S1, RRS1, PDIA6, PLEC, TEBP, NONO, PCBP1, PCBP2, DHC24, SF3B3, SF3A1, TRAM1, ELAV1, AAAT, RBBP7, H31T, PDS5A, TSR1, IF2GL, RRP12, NU188, HP1B3, EF1A3, PPR18, PRP8, C1TM, DHX30, CAND1, MISP, SPB1, PELP1, RDH10, CCAR2, TXND5, STT3B, BRX1, PO210, GEMI5, RT27, HS105, GCN1, NU205, AKAP1, AN32B, RBP56, DDX17, FUBP2, TNPO1, UBP7, UTP4, LRC59, PGAM5, FUBP3, MBOA7, MCCA, WRIP1, UHRF1, POP1, HCD2, ROAA, TM9S2, TCPH, ANM1, H2B1L, RNZ2, MEP50, MBB1A, ESYT1, H2AJ, GNL3, HDHD5, GTPB4, API5, RPF2, SFXN1, RDH14, ABCB6, DDX21, MDN1, DCA13, ATD3A, DDX18, MIC19, TEX10, TECR, MYOF, THYN1, HACD3, RRBP1, ABC3B, RLP24, ACINU, OGDHL, COR1C, PRP19, SSRG, TRI33, EIF3L, RUVB1, VDAC3, PDIP2, NOP58, SF3B6, RTCB, RL36, LAS1L, SRPRB, COPG1, MTCH2, CEPT1, ZNT1
Species: Homo sapiens
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Ramirez DH, Yang B, D'Souza AK, Shen D, Woo CM. Truncation of the TPR domain of OGT alters substrate and glycosite selection. Analytical and bioanalytical chemistry 2021 34725712
Abstract:
O-GlcNAc transferase (OGT) is an essential enzyme that installs O-linked N-acetylglucosamine (O-GlcNAc) to thousands of protein substrates. OGT and its isoforms select from these substrates through the tetratricopeptide repeat (TPR) domain, yet the impact of truncations to the TPR domain on substrate and glycosite selection is unresolved. Here, we report the effects of iterative truncations to the TPR domain of OGT on substrate and glycosite selection with the model protein GFP-JunB and the surrounding O-GlcNAc proteome in U2OS cells. Iterative truncation of the TPR domain of OGT maintains glycosyltransferase activity but alters subcellular localization of OGT in cells. The glycoproteome and glycosites modified by four OGT TPR isoforms were examined on the whole proteome and a single target protein, GFP-JunB. We found the greatest changes in O-GlcNAc on proteins associated with mRNA splicing processes and that the first four TPRs of the canonical nucleocytoplasmic OGT had the broadest substrate scope. Subsequent glycosite analysis revealed that alteration to the last four TPRs corresponded to the greatest shift in the resulting O-GlcNAc consensus sequence. This dataset provides a foundation to analyze how perturbations to the TPR domain and expression of OGT isoforms affect the glycosylation of substrates, which will be critical for future efforts in protein engineering of OGT, the biology of OGT isoforms, and diseases associated with the TPR domain of OGT.
O-GlcNAc proteins:
UBA6, SBNO1, CNOT1, SMHD1, ADAS, DX39A, BACH, MYO1C, PSD11, PSD12, TAF4, CLIC1, EIF3F, IPO5, DNM1L, AGRIN, PLOD2, IMA4, NOP56, DDX3X, PDXK, ANM5, TCRG1, PSA7, SCAM3, HAT1, HGS, MYPT1, HNRDL, XPO1, PUR4, NPC1, SCAM2, ARC1B, ARPC2, ARPC3, RPAC1, GNPAT, ZN185, SURF4, OGT1, PPM1G, EIF3D, EIF3H, P4HA2, MAGB2, NUP42, DHX15, MCES, SERA, PSMD3, PAPS1, ZW10, HNRPR, TXNL1, E41L2, XPOT, TIM44, PLRG1, ZN207, GET3, BUB3, ACTN4, AP1G1, SNG1, SNG2, SYNC, MYO1B, NRDC, CALU, EDIL3, SAHH2, DHX16, SMCA5, KDM1A, OGA, HNRPQ, PLOD3, DIAP1, TSN3, PLIN3, CTND1, MAGC1, USO1, DKC1, IF2P, DNJA2, WDR1, CPNE3, T22D2, ANR17, H2AY, FLNB, CISY, SC22B, PR40A, ERLN1, SF3B1, CSDE1, U520, NU155, EIF3G, FACE1, IDHC, ATRN, STAM2, PRAF3, FLOT1, RL1D1, WFS1, MTA2, TOX4, SC24D, SUN1, FRYL, NFAT5, GLSK, SC31A, UBR5, LC7L3, FKBP9, SMC2, IPO7, AHSA1, SGPL1, SC24B, ACSL3, CDS2, AP2A1, BAG2, AIFM1, FADS2, CLPT1, TOM40, ACL6A, LDHA, COX2, PNPH, AATM, PGK1, ASSY, FOS, LMNA, FINC, TFR1, NU4M, NU5M, GLCM, FUCO, ALDOA, ANXA1, OAT, TBB4A, G3P, CPNS1, HSPB1, TYSY, RPN1, RPN2, GNAI2, AT1A1, AT1B1, ALDH2, ADT2, PCCA, IF2A, RLA2, RLA0, LA, ITB1, ATPB, IF4E, ENOA, G6PI, NPM, ITAV, HYEP, LDHB, PDIA1, H10, CATD, ANXA2, CAN1, TBB5, TRY1, SAP, PROF1, SYEP, CATB, HS90A, HNRPC, LAMB1, YES, FUMH, TSP1, ANXA6, MDR1, 4F2, PFKAM, HS90B, SRPRA, ASNS, ODPA, RU17, GNAI3, ANXA5, RSSA, CD63, SNRPA, GSTP1, LEG1, CN37, DLDH, ROA1, PARP1, LKHA4, ALDOC, GPHRB, HS71B, LYAG, RRAS, H14, ODP2, THIO, ESTD, CH60, CLUS, BIP, LAMC1, PPAL, HSP7C, GTR1, GTR3, ODPB, PYGB, LAMP1, TOP1, TOP2A, G6PD, PYC, C1TC, MPRI, ADHX, PABP1, PCNA, HARS1, IMDH2, TPR, KCRB, ACTN1, PEPD, XRCC6, XRCC5, LAMP2, RINI, EF2, PDIA4, P4HA1, TCTP, ACPH, ETFA, GLU2B, FPPS, KPYM, ENPL, IDE, HNRPL, SYDC, ALDR, B4GT1, EZRI, FOSL2, NDKA, GNS, RS2, RFA2, BGAL, H12, AT2A2, FAAA, AATC, JUNB, ITA2, CAN2, PYRG1, DDX5, PFKAL, PRS6A, TCPA, RL35A, ARF4, RL7, VINC, SON, RL17, PGAM1, DNLI1, PLCG1, NUCL, HXK1, RPB3, E2AK2, TFE3, SPEE, IF2B, ANXA7, PSB1, MPRD, LMNB1, VATB2, FLNA, VDAC1, TGM2, FBRL, PUR2, PUR6, UBA1, NDKB, ADRO, ROA2, RFX1, QCR2, SFPQ, PPIB, SYWC, RS3, SAHH, COF1, IF4B, PPAC, RPB1, MCM3, RS12, DNJB1, ATPA, PSA1, PSA2, PSA3, PSA4, ITA3, MOES, DDX6, DNMT1, U2AF2, RL13, PTBP1, SYTC, SYVC, EF1G, STOM, RL10, RFA1, APEX1, CD82, PYR1, CALR, MAP4, CALX, PSB8, PSA5, PSB4, PSB6, PSB5, DPOD1, TEAD1, MK01, AMPL, IMPA1, EPHA2, TKT, EF1D, PRDX6, BLVRB, PRDX5, PRDX3, RL12, ECHM, PEBP1, PDIA3, 2AAA, CDC27, PURA2, AMRP, PUR8, CLIP1, SL7A1, RPB2, SDHA, GDIA, METK2, DNJA1, QCR1, PUR9, HNRH3, HNRH1, 1433B, 1433S, STIP1, PRDX2, RL9, KINH, MRP1, MCM4, MCM5, MCM7, GLYM, HSP74, CTNA1, CTNB1, PHB1, RADI, RFC4, RL22, FBN2, GDE, MYH9, MYH10, COPB2, SOAT1, ADDA, BASI, FUS, NU214, PRS7, MP2K2, ATPG, HEM6, RL4, PGM1, ODO2, SRP14, NUP62, FDFT, TAGL2, TALDO, RBMX, VKGC, VATA, GRP75, IF4A3, RS19, RL3, OST48, AN32A, CAP2, TCPZ, RL13A, STAT3, MDHC, MDHM, ECHA, IF2G, KDM5C, GARS, SYIC, STAT1, CASP3, LPPRC, SATT, LIS1, MUC18, MATR3, MSH2, MAGA4, RANG, NAMPT, PRS6B, PPIC, VDAC2, UBP5, KI67, RAGP1, RECQ1, NOP2, CRKL, BAG6, NSF, RL27A, RL5, RL21, RL28, RS9, RS5, MAP1B, UTRN, IQGA1, STT3A, CAPZB, SYQ, UCRI, PPCE, COPD, LSS, CD151, PSMD8, TCPE, NEST, HSP13, PIPNB, PAXI, AL9A1, RL34, LMAN1, NASP, FAS, TCPG, EFTU, AL7A1, SYAC, SYCC, SYSC, PRI2, PSB3, PSB2, MCM2, ACADV, YLPM1, TMEDA, RBM25, NU153, RBP2, NDUV1, GSK3A, TAF6, GUAA, GDIB, EMD, SERPH, VASP, TCPQ, TCPD, ANX11, PAPOA, FXR1, RAB5C, RAB7A, RT29, SMCA4, BAP31, HCFC1, DHB4, HDGF, ROA3, 6PGD, HNRPM, IMA1, GDIR1, AGFG1, HNRPF, MSH6, RBM5, SPSY, CAZA1, NUP98, BIEA, PPP5, ACLY, MAP11, COPB, COPA, SC5A3, SMTN, MOT1, IST1, SC24C, SYRC, SYYC, UBP14, BCAT1, AT1B3, RD23B, P5CS, IF5, S12A2, XPO2, TERA, ECHB, AFAD, NP1L1, DSRAD, SEC13, PSA, HNRH2, EIF3B, ATPK, SYMC, IF6, CTBP2, TMM33, GEMI4, CORO7, NU107, ARPC4, TPIS, EIF3E, PP4C, IF4A1, PSA6, CDC42, DEST, GMFB, SRP54, RAB10, UBC12, ARP3, ARP2, ABCE1, RAP2B, RS3A, RL15, RL37A, S61A1, DAD1, NPC2, HNRPK, 1433G, RS7, PRS4, PRS8, RS8, RS15A, RS16, 1433E, RS14, RS23, RS13, RS11, SMD2, SMD3, PRS10, RL7A, ERF1, CNBP, RS4X, PP2AB, RS6, H4, RAB1A, RAN, RL23, RS25, RS28, RL30, RL31, RL10A, RL32, RL11, RL8, PPIA, TRA2B, AP2B1, 1433Z, IF5A1, RACK1, YBOX1, UB2L3, TBA4A, IF4G2, GTF2I, TCPB, PRKDC, RL24, SRSF3, FOXK1, DAB2, RBM10, MPCP, VIGLN, CLH1, FKBP3, HNRPU, U2AF1, SPTB2, AMPD2, CAP1, HMCS1, LAT1, EXOSX, PFKAP, EWS, PLCB3, ODO1, RL18A, MP2K1, FKBP4, PLOD1, RL6, TOP2B, DYST, KMT2A, LMNB2, CEBPZ, TF65, IF4G1, LGUL, 1433F, DYN1, EF1A2, PTN11, PUR1, GFPT1, EXOS9, SUH, GABPA, PRDX1, RL18, C1QBP, CKAP4, KHDR1, LRP1, SRSF1, PP2BA, DHX9, GOGA2, LG3BP, CD47, SSRP1, NSUN2, NCBP1, EP300, AHNK, MGAT2, GALT2, AP1B1, MPPA, SCRN1, NU160, SCAP, TBL3, TF3C1, ASPH, TROP, BPTF, AIMP1, ILF2, ILF3, LMAN2, TRAP1, FLII, ACACA, CSN1, MTAP, EI2BE, TADBP, ROA0, PRDX4, PSMD2, SRSF6, TIF1B, DHRS2, G3BP1, MTA1, EIF3I, UB2V1, DC1I2, NNTM, TCOF, TMED1, VPP3, PICAL, RIPK1, CUL2, CUL4A, CUL4B, FCL, MOGS, CD166, SPTN1, DX39B, BLMH, CKAP5, COTL1, HNRPD, SCRB2, EIF3A, UBP2L, SCRIB, FHL2, DPYL3, DCTN1, DYHC1, SRC8, FLOT2, TRI25, FLNC, GALE, CAPR1, RBM39, MCM6, TRIPC, PUM1, EPN4, RRP5, BMS1, UBP10, GANAB, LBR, MVP, MEF2D, CHD4, LASP1, GPNMB, ZN638, IMB1, NUMA1, CND2, SPCS2, PSMD6, ABRX2, SEPT2, CND1, U5S1, SYK, WDR43, PDIA6, PA1B3, IPYR, TEBP, NONO, PWP2, RCN1, L2GL1, TMED2, PCBP1, PCBP2, SF3B3, SC23A, SF3A1, TSN, SF01, PCH2, MED1, MARE1, ELAV1, NSDHL, TAB1, AAAT, MLF2, VAS1, ZYX, SEPT7, ADRM1, CCDC6, UAP1, PSMD5, PKN2, DDB1, DPYL2, KCC4, SRSF7, DREB, NRF1, FSCN1, IF16, PCKGM, UPP1, UGPA, TRXR1, HNRL2, INF2, PDS5A, QSER1, QRIC1, SMU1, P3H1, K2C71, TIM50, GXLT1, TB10B, SNUT2, HERC4, P4R3B, EOGT, CE170, UBR4, SKT, UBAP2, RBM26, RPRD2, ZMYM4, TWF2, LIN54, RHBD2, MBOA5, EDC4, PRP8, SCYL2, NFRKB, TTC37, NCEH1, BRAT1, ZC3HE, C1TM, SBSN, CREL2, PACS1, GGYF2, SRCAP, TMTC3, IKIP, SPT6H, SND1, DDX46, BZW1, DHX30, EIF3M, CYFP1, KDM3B, RIR2B, ERMP1, ZCCHV, NUP54, POGZ, MYH14, CLAP1, EMSY, SH3R1, HUWE1, TMED4, SYVN1, ZN598, PHLB1, LUZP1, CAND1, TSYL5, EZHIP, CARM1, DDX42, P66A, C2CD5, I2BP1, RB6I2, PLD3, P3H3, WDR75, Z3H7A, ANKH1, CCAR1, DJC10, SMAP1, SPART, RPTOR, NUP93, ZN687, SYNPO, FNBP4, CPSF7, EMC1, ENAH, S43A3, GT251, SUMF2, PGLT1, TXND5, PAIRB, LS14A, ABCF1, FBX22, NEUA, CADM4, ATLA2, PLBL2, WDR36, SMRC2, NPL4, S35B2, UBA3, TBC15, CIP2A, STT3B, PNPT1, HM13, DTX3L, PO210, GEMI5, PARD3, IPO4, SCRB1, RAB2B, NU133, PDC6I, S20A1, ATX2L, PALLD, DDX1, GBF1, NICA, TM9S4, HS105, GCN1, PRP16, NU205, PXDN, TFG, ARC1A, HDAC2, CBP, SYMPK, DDX17, CELF1, UFD1, GSLG1, RENT1, TNPO1, GLMN, USP9X, UBP7, SCAM4, SMCE1, MYDGF, PIGT, NCLN, EXOC4, FERM2, FUBP1, FKB10, APH1A, PPWD1, AP2M1, RCN3, ELP4, SEH1, DNJA3, CYFP2, PGM2, PDLI5, ERO1A, FUBP3, ITCH, TMX3, CNDP2, ZFR, EP400, SNX27, PRRC1, ZN512, MBOA7, DOCK7, ARAP1, IPO9, RBM14, NED4L, VPS35, CIC, MED15, EFGM, MCCA, MYADM, SIN3A, MINT, PNKP, NIBA2, TBCB, PSB7, CNN2, PSMD1, EYA3, SORT, NUP88, P2RX4, DNJC7, PHB2, KIF2C, ATX2, HCD2, ROAA, NP1L4, ACON, TM9S2, S29A1, TS101, TCPH, ANM1, CND3, MBB1A, COR1B, GCP2, ESYT1, DIDO1, MCMBP, TBCD, TBB6, RPC3, HNRL1, HTAI2, TMED9, NUP58, SFXN3, OSB10, NAA15, S12A9, SRRT, EIF2A, WDR11, FTO, TTYH3, WDR12, YIPF3, PITH1, PAIP1, NAT10, XRN2, TOLIP, SP130, BRD8, DDX47, MAGT1, RAB1B, LMA2L, I2BPL, GHITM, TMX1, PTN23, OFUT1, PIGU, WNK1, AMPB, HEAT1, NOL6, WDR26, AT133, ACAD9, GORS2, MARH7, SFXN1, PKHA5, XPO5, MYG1, EMAL4, TM9S3, APMAP, ARFG3, RTN4, ANLN, XPP1, PDLI7, DDX21, EI2BG, MBNL1, HEBP1, SERC1, F1142, SYFB, SYIM, SMC4, OLA1, RBM12, T106B, CYRIB, DCA13, CC50A, SEP11, FANCI, ARL8B, DDX18, TM160, MIO, CARF, DPP3, RM39, UGGG1, ERAP1, HPBP1, MAT2B, MYOF, ITSN2, HACD3, SEP10, RCC2, DIP2B, CT2NL, RRBP1, SYLC, RBM27, SUCB1, MRC2, DHCR7, DJB11, SAE2, NXF1, SUN2, SRP68, SEPT9, UBQL2, PCYOX, CEIP2, S30BP, MED13, PUF60, NRBP, VATH, IPO11, XPO7, BAZ2A, GGT7, NAGK, DBNL, CDC23, STML2, PARP4, NUDT5, TF3C4, ACINU, RAB21, DNPEP, ASAP1, MRTFB, MED23, HECD1, PKCB1, COR1C, MYO6, PRP19, MAGD2, PSD13, MINP1, NOVA2, WDR3, S12A4, MACF1, SCAF8, TRI33, SMAG1, XCT, SRRM2, PA2G4, SMC3, RUVB2, EIF3L, PLAP, RUVB1, VDAC3, ERGI3, SC5A6, DRG1, NCKP1, AT11B, E41L3, CHSP1, NOP58, ZN281, SYYM, TMX2, SHLB1, SBDS, TMED7, STRAP, RTCB, FBX7, RBGP1, NOC2L, TLN1, DCAF1, HYOU1, PRC2C, YTHD2, SP16H, TNPO3, MAGD1, SNX8, TRUA, NPTN, S12A7, COPG1, BIG1, BZW2, DC1L1, IF2B2, ZNT1, S4A7, ZHX2, S23IP
Species: Homo sapiens
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Zhang W, Liu T, Dong H, Bai H, Tian F, Shi Z, Chen M, Wang J, Qin W, Qian X. Synthesis of a Highly Azide-Reactive and Thermosensitive Biofunctional Reagent for Efficient Enrichment and Large-Scale Identification of O-GlcNAc Proteins by Mass Spectrometry. Analytical chemistry 2017 89(11) 28510447
Abstract:
O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification of proteins in eukaryotic cells. Despite their low abundance, O-GlcNAc-modified proteins play many important roles in regulating gene expression, signal transduction, and cell cycle. Aberrant O-GlcNAc proteins are correlated with many major human diseases, such as Alzheimer's disease, diabetes, and cancer. Because of the extremely low stoichiometry of O-GlcNAc proteins, enrichment is required before mass spectrometry analysis for large-scale identification and in-depth understanding of their cellular function. In this work, we designed and synthesized a novel thermosensitive immobilized triarylphosphine reagent as a convenient tool for efficient enrichment of azide-labeled O-GlcNAc proteins from complex biological samples. Immobilization of triarylphosphine on highly water-soluble thermosensitive polymer largely increases its solubility and reactivity in aqueous solution. As a result, facilitated coupling is achieved between triarylphosphine and azide-labeled O-GlcNAc proteins via Staudinger ligation, due to the increased triarylphosphine concentration, reduced interfacial mass transfer resistance, and steric hindrance in homogeneous reaction. Furthermore, solubility of the polymer from complete dissolution to full precipitation can be easily controlled by simply adjusting the environmental temperature. Therefore, facile sample recovery can be achieved by increasing the temperature to precipitate the polymer-O-GlcNAc protein conjugates from solution. This novel immobilized triarylphosphine reagent enables efficient enrichment and sensitive detection of more than 1700 potential O-GlcNAc proteins from HeLa cell using mass spectrometry, demonstrating its potential as a general strategy for low-abundance target enrichment.