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Hou C, Wu C, Wu Z, Cheng Y, Li W, Sun H, Ma J. Systematic Evaluation of Affinity Enrichment Methods for O-GlcNAc Proteomics. Journal of proteome research 2024 39302247
Abstract:
O-Linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) on proteins plays critical roles in the regulation of diverse biological processes. However, protein O-GlcNAcylation analysis, especially at a large scale, has been a challenge. So far, a number of enrichment materials and methods have been developed for site-specific O-GlcNAc proteomics in different biological settings. Despite the presence of multiple methods, their performance for the O-GlcNAc proteomics is largely unclear. In this work, by using the lysates of PANC-1 cells (a pancreatic cancer cell line), we provided a head-to-head comparison of three affinity enrichment methods and materials (i.e., antibody, lectin AANL6, and an OGA mutant) for site-specific O-GlcNAc proteomics. The enriched peptides were analyzed by HCD product-dependent EThcD (i.e., HCD-pd-EThcD) mass spectrometry. The resulting data files were processed by three different data analysis packages (i.e., Sequest HT, Byonic, and FragPipe). Our data suggest that each method captures a subpopulation of the O-GlcNAc proteins. Besides the enrichment methods, we also observe complementarity between the different data analysis tools. Thus, combining different approaches holds promise for enhanced coverage of O-GlcNAc proteomics.
O-GlcNAc proteins:
NBAS, SBNO1, PRS37, CNOT1, MGT4D, P121C, P20L1, MCRI1, SPT5H, MANBA, AGRIN, BCL9, NOP56, ARI1A, ABLM1, KMT2D, SCAM3, MYPT1, HNRDL, XPO1, SYN3, SC16A, SYNJ2, SYNEM, TIF1A, LAMA5, MCES, DC1L2, CP110, IF4G3, E41L2, ZN207, BUB1, HTSF1, AKAP8, DHX16, MGRN1, TRAK2, KIF1B, PLIN3, MAFK, IF2P, DTNB, MITF, SRGP2, ATG13, ZC11A, PP6R2, ANR17, LRP5, NDUS2, NCOR1, U520, UTP20, WDHD1, FACE1, TOX4, SC24D, SUN1, SC31A, CE152, UBR5, AGFG2, APOL3, IPO7, SVIL, SC24A, SC24B, ZMYM6, GPR75, LATS1, LMNA, GCR, OAT, CPNS1, HSPB1, GNAI2, A4, K1C18, ATPB, SAP, PROF1, HS90A, HS90B, NFIC, VIME, SNRPA, ATX1L, DERPC, PTPRF, ESTD, ODPB, C1TC, SKIL, XRCC6, CCNB1, ATF2, NDKA, GNS, ZEP1, AT2A2, ATF7, ERCC2, EGR1, ATF1, GGT1, AT2B1, FLNA, 5NTD, PUR2, RFX1, SFPQ, RS3, STOM, ARNT, MAP4, KPYR, CLIP1, HXC9, ZEP2, QCR1, I5P2, ELF1, MRP1, TRY3, CAH8, T2FA, NU214, NUP62, STAT3, ECHA, HNF4A, LAP2A, LAP2B, CACP, GATA4, BAG6, MAP1B, STT3A, RFX5, PAXI, LMAN1, CDK8, CENPF, YLPM1, NU153, RBP2, GSK3A, TAF6, DNLI3, EMD, TCPD, PAPOA, HNRPM, AGFG1, STAT2, NUP98, ATX1, ATN1, RD23B, AF10, BRPF1, NP1L1, EIF3B, MAZ, HDAC4, IF6, H2B1D, ACTB, ABCE1, RRAS2, ERF1, RL32, ACTG, EF1A1, CSK21, F193A, SARNP, FOXK1, ZBED6, RHG04, CLH1, SPTB2, TIAR, SP2, DYST, ATP7A, NOTC2, TLE4, PTN12, FAK1, ZO1, ACK1, DHX9, EP300, AHNK, NEXN, CPSF1, WASC5, TROAP, BPTF, SF3A3, TP53B, PTN13, TRAP1, DLG1, ATM, PPIL2, TCOF, SMAD4, PICAL, MAMD1, ASPP2, RIN1, CDK13, VEZF1, UBP2L, SRC8, CAPR1, ITPR1, MDC1, RRP1B, UBP10, COG2, MEF2D, LASP1, IMB1, PLEC, NONO, SF3A1, JHD2C, ELF2, TAB1, NCOA1, ZYX, UAP1, DREB, PDS5A, QSER1, TXIP1, FHOD3, P3H1, EMAL3, ZN791, CHD9, TB10B, FND3B, CRTC2, BCORL, BRD10, CE170, ZN362, ZEP3, UBR4, UBAP2, AHDC1, RPRD2, ZN318, TUT7, RN213, TENS2, WAC2A, MAP1S, ARID2, TENS3, ANR40, BICRL, ATLA3, CPIN1, GRAM4, NIPBL, LIN54, RINT1, L2GL2, C2D1A, SCYL2, NFRKB, ZC3HE, LARP1, ZNT9, CRTC3, GLB1L, MCAF1, SRCAP, CFA65, UBN2, VP13C, RAPH1, H32, TMUB2, MARK2, DDX46, EIF3M, KDM3B, GRIN1, KANL1, POGZ, NUFP2, MAVS, DHX29, EMSY, RAI1, I2BP2, HUWE1, YTHD3, KAISO, MYPN, LYRIC, KTN1, PRSR1, DDX42, P66A, RHPN2, NUDC3, AHNK2, GID4, ARI3B, MA7D3, MGAP, LMTK2, ANKH1, SUGP1, MILK2, SRRM1, PHAR4, DCP1B, PELP1, TENS4, CCAR2, HROB, TXND5, RB15B, TNR6A, PHC3, NAV1, VP37A, SYNE1, FAD1, NUP35, TOIP2, KNL1, O52N2, TRY6, COP1, GOGA5, ALMS1, DLG5, MORC4, GEMI5, PIGO, PARD3, IPO4, TM263, PDC6I, LMO7, ATX2L, PHIP, P66B, BBX, SMG7, F168A, PRCC, RYR2, US6NL, SYMPK, RENT1, PF21A, EFHD2, KBP, F120B, RBM33, THOC1, P121A, PDLI5, VPS50, DNJC1, Z512B, ZFR, EP400, SENP8, PRRC1, DOCK7, RBM14, NEK1, PP1RA, PHF12, SPAG5, CIC, ERBIN, SIN3A, MINT, HTF4, VAT1, PLIN2, DNJC2, MNT, KIF2C, ATX2, ARI3A, GDF15, WAC, DIDO1, RFIP5, SRRT, SETD2, ABCA2, TRIM5, TANC1, ZN407, SP130, PUS7L, I2BPL, NELFA, CHM4B, WNK1, ZHX3, CDCP1, Z385D, BCAS3, PEAK1, ECT2, JUPI2, ZN408, PKHA1, ILRL2, RC3H2, TAF9B, NCOA5, TANC2, SYTL2, CHD8, UBN1, DCP1A, SEM4B, HPS4, PDLI7, 4ET, CHRC1, F1142, LTBP3, SYIM, IGSF5, RBM12, STAU2, TYDP1, SLTM, CARF, HXC10, TAB2, CDK12, ERAP1, MYOF, BICRA, CNOT2, SPN90, F135A, RBM27, KANL3, ZMYM2, LIMD1, TCF20, UBQL2, S30BP, PUF60, SIX4, TRM6, NOCT, TASOR, GMEB2, ACINU, CNO11, ASAP1, YETS2, ZMYD8, MYO6, SPAT2, PRP19, NSF1C, MACF1, TRAK1, SRRM2, SCML2, PDE10, AP3M1, ZN281, ZN711, TLN1, ARIP4, DCAF1, WAC2C, RPGF2, CRBG1, PRC2C, UTP18, SNX9, NCOR2, BIG1, NCOA3, PCLO, S23IP
Species: Homo sapiens
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Shen B, Zhang W, Shi Z, Tian F, Deng Y, Sun C, Wang G, Qin W, Qian X. A novel strategy for global mapping of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry identification. Talanta 2017 169 28411811
Abstract:
O-GlcNAcylation is a kind of dynamic O-linked glycosylation of nucleocytoplasmic and mitochondrial proteins. It serves as a major nutrient sensor to regulate numerous biological processes including transcriptional regulation, cell metabolism, cellular signaling, and protein degradation. Dysregulation of cellular O-GlcNAcylated levels contributes to the etiologies of many diseases such as diabetes, neurodegenerative disease and cancer. However, deeper insight into the biological mechanism of O-GlcNAcylation is hampered by its extremely low stoichiometry and the lack of efficient enrichment approaches for large-scale identification by mass spectrometry. Herein, we developed a novel strategy for the global identification of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry analysis. Standard O-GlcNAc peptides can be efficiently enriched even in the presence of 500-fold more abundant non-O-GlcNAc peptides and identified by mass spectrometry with a low nanogram detection sensitivity. This strategy successfully achieved the first large-scale enrichment and characterization of O-GlcNAc proteins and peptides in human urine. A total of 474 O-GlcNAc peptides corresponding to 457 O-GlcNAc proteins were identified by mass spectrometry analysis, which is at least three times more than that obtained by commonly used enrichment methods. A large number of unreported O-GlcNAc proteins related to cell cycle, biological regulation, metabolic and developmental process were found in our data. The above results demonstrated that this novel strategy is highly efficient in the global enrichment and identification of O-GlcNAc peptides. These data provide new insights into the biological function of O-GlcNAcylation in human urine, which is correlated with the physiological states and pathological changes of human body and therefore indicate the potential of this strategy for biomarker discovery from human urine.
O-GlcNAc proteins:
TX13C, ESYT2, BICL2, ODAM, SRCRL, SYTC2, Z804B, O2A25, XIRP2, NKX26, SMCO2, MFS2B, O51F1, NCF1B, ABTB3, SRRM4, D11L8, YV023, LEUTX, MEIOS, RB27B, CACB4, SOCS6, CHD1, NDC80, KIF3C, MYPT1, IPP2C, MUSK, MRP3, PA2GX, ARPC5, P2RX6, ZW10, ZN749, KCAB3, ACTN4, VEGFD, BNI3L, ZN292, PI51C, MABP1, CCG3, NOBOX, REV3L, TBL1X, NBN, KI21B, PX11A, UBP2, CAN15, ATRN, SPF30, MYO1D, SUN1, ENDD1, M4K4, CFAB, LV151, K1C14, K2C1, HSPB1, CHLE, SAP, SRPRA, GNAI3, IL6RA, VILI, AT8OS, GLI3, RNAS2, LYAG, SPTB1, PSG2, LAMP2, CSPG2, SC5A1, F261, DPEP1, EPB42, ITB6, LMNB1, NEBU, RYR1, TENX, SP100, ANX13, ADH6, GNA11, NMT1, CPSM, GBRA5, AKT2, I5P2, MYH11, HMGCL, PGM1, CDN1A, MLH1, SATT, DCC, MAGAA, MMP13, ABCG1, MP2K3, UTRN, IDHP, PSB3, RBP2, MRE11, ETV1, IRAK1, ARSD, NEK4, SPSY, COPA, SMTN, ATN1, PMS1, PMS2, ATNG, PRRX1, AF17, NXPH1, NAL12, IF4A1, STX1B, KCNJ2, HPCA, PKD1, REEP5, CAC1D, MMSA, SEMG2, ACY1, P, ZNF91, LG3BP, PDE4C, SCAP, PO4F2, NMDE1, OVGP1, ANK3, NFAC3, AKAP6, CENPR, SF3B2, TRA2A, CUL4B, ALKB1, CO9A2, FA53B, WRN, SLBP, GOGB1, ZN169, ODFP1, FRPD4, MELT, SART3, IF4H, KIF14, PLCL1, PLEC, PSG5, DLGP5, MARE2, ENOX2, CNGA2, AINX, HCDH, CSPP1, QSER1, ZN423, SPKAP, PRSR3, ATOSA, K2C71, GON4L, GNPTA, LEGL, PAR14, PCDP1, PLCH1, AARD, RHG29, SPIN4, FA76B, CX066, BRM1L, ODR4, SZT2, SYRM, EEIG2, CE162, CL060, MYOME, ARHGG, CA140, CARL1, ZMYM4, EST4A, F219B, WDR25, F90A2, LAR1B, ATG9B, ARID2, FTM, USF3, KCD16, ZNF57, K1C39, Z518A, BLT3A, CV042, RTL6, CAPR2, ADAM5, ZNT6, CA094, VIP1, AGRD1, CC171, KLH24, ABRX1, PAMR1, TM14E, ITIH6, RFIP1, IGS10, WDR87, SHSA6, FGD6, RGMC, NEK10, UBP31, NOL8, MARK2, BEND5, PCAT2, EPMIP, DPH6, OLIG3, TRPM8, CC186, MYCPP, TMC7, Z804A, TAF8, RALYL, RBM23, CC190, PKHL1, GA2L3, TCAM2, STX12, KI18B, RB6I2, ARMC8, K0825, STH, RP1L1, TPH2, F217A, PLPL6, EFC13, CJ067, PSYR, TBC21, DOCK3, AGRF4, SYCE1, CADM3, CP4Z2, CLASR, ZN786, CLIP4, CBPA6, NKAP, TM156, CPSF7, EFNMT, IGS22, LMBL4, ZFP62, PHC3, DDHD1, EXPH5, NAV1, BD1L1, BPIB6, TET1, MYRIP, FBH1, O10K1, ST3L4, CHD6, DMXL2, MIPT3, ES8L3, DOT1L, NAT14, CKAP2, ARAP3, CSKI1, ATRIP, MUC16, ELYS, TITIN, LZIC, PHF3, TBCD5, K0232, PRCC, TFG, SFRP3, COR2A, ARHG2, TATD2, LENG9, FAM3D, ALKB8, CHM4C, LRC58, REPS1, PGBD4, P3IP1, CDCA5, HMCES, DMAC1, IGS21, PAWR, ITCH, M4A14, CLMN, S41A2, HSH2D, TM87B, ZN514, P12L2, C295L, CQ10A, ROBO3, WWAS2, DB118, KI20B, PHF12, SPAG5, OR2M4, HMCN1, RANB9, CCNL2, IWS1, K1C12, NPY6R, S1PR3, LYST, CDC6, EBP2, NIPS1, DDX50, FA83C, PIMRE, NDC1, MTNA, UT14A, NUP85, TDRD1, MSTRO, SETD2, OSBL8, UACA, TSG10, TB182, EGLN1, CRLD2, CSTFT, NKX24, CT191, ESF1, PIEZ2, RANB3, CSR2B, UCK1, ZN556, MLXIP, TNR6C, HMX1, EQTN, PRDM9, TLR8, HELLS, TOPRS, KIF15, RAD18, HOME2, BRWD1, TEN2, CCM2L, FGOP2, MCUB, MIC19, KCTD5, CARF, FAT2, DTL, SACS, KLHL8, CE126, WDR35, NRX2A, DNM3B, COPG2, GCP4, PARP2, TCF20, ASIC3, RABX5, STAG3, NGAP, FBX5, MKLN1, ZBT21, PKHH1, SOX13, LIMC1, EXOC7, CBPC1, TUTLB, XCT, SHOC2, RUVB2, PDE10, PRLD1, FBW1A, DLGP4, WDR37, ZBTB1, NSG2, LSM2, DOP2, C170B, SAM50, PCDBB, PCDA3, TF3C3, CCG2, BIG1, S4A4, PCLO
Species: Homo sapiens
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