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Burt RA, Dejanovic B, Peckham HJ, Lee KA, Li X, Ounadjela JR, Rao A, Malaker SA, Carr SA, Myers SA. Novel Antibodies for the Simple and Efficient Enrichment of Native O-GlcNAc Modified Peptides. Molecular & cellular proteomics : MCP 2021 20 34678516
Abstract:
Antibodies against posttranslational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of these PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1300 unique O-GlcNAc-modified peptides and over 1000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.
O-GlcNAc proteins:
IQIP1, A0A0A6YWG7, A0A0G2JF55, A0A0N4SW93, A0A0R4J060, A0A0U1RPL0, A0A140LIW3, A0A140T8K9, A0A1B0GS41, A0A1B0GS91, A0A1D5RMI8, A0A1L1M1J8, A0A1L1SR84, A0A1N9NPH8, A0A1Y7VNZ6, A0A286YDB3, A0JNY3, A2A482, A2A654, TANC2, LZTS3, AJM1, BCORL, A2AUD5, A2AWN8, B1ASA5, B1ATC3, B1AUX2, B2RQL0, CSPP1, B2RY58, B7ZNA5, CTTB2, D3YU22, D3YUV1, D3YWX2, D3YZ21, SHAN1, D3Z5K8, E0CXZ9, E9PUL3, PRRT2, E9PUR0, E9PV26, E9PVY8, SET1A, E9Q0N0, E9Q3E2, E9Q3G8, E9Q4K0, ARI1B, SETD2, E9Q6H8, E9Q6L9, E9Q828, E9Q9C0, E9Q9Y4, E9QAQ7, E9QAU4, E9QAU9, E9QKI2, E9QLZ9, E9QM77, F2Z3U3, F6RQA2, SYGP1, F7C376, BICRA, F8VQL9, F8WIS9, G3UZM1, G3X8R8, G3X928, RFIP2, H3BKF3, H3BKP8, H9KV00, J3QNT7, DPYL2, PRDX6, MNT, NUMBL, PEX5, BMPR2, CTND2, PITM1, ACK1, CAC1B, SYUA, DSG2, SPT5H, E41L2, SP3, KDM6A, CPNS1, ZFR, HCN1, CTBP1, BSN, STAM2, SYN1, MBP, EGR1, NFL, NFM, ITB1, RC3H2, ATX1L, RL7A, MAP1B, VIME, EIF3A, RGRF1, PABP1, FOXK1, EAA2, CBP, RFX1, SOX2, KPYM, CTBP2, GCP3, TB182, GMEB2, PI5PA, DOCK4, PCBP1, LIPA3, RS3, PAX6, KCNJ3, PP2BA, TBA4A, STAM1, NCOA1, CXB6, WNK1, PSME2, WBP2, SHPS1, NRSN1, CTNB1, PLAK, S30BP, NFIA, ZEP1, YES, CAPR2, MITF, GRD2I, Q0VF59, HDX, MA6D1, F171B, ZFHX2, MLXIP, PDLI7, PRC2C, CIART, YETS2, SRBP2, Q3U2K8, GSE1, RREB1, WNK2, DAB2P, ZEP2, AAK1, TNR6A, GRIN1, SRBS2, GRM5, Q3UZG4, RBM44, Q3ZB57, PHAR4, RESF1, Q5EBP8, UNKL, VP13A, COBL, KDM6B, PRSR1, Q5RIM6, SMG7, RBM27, TM1L2, Q5SVJ0, Q5SXC4, SIN3A, GAS7, CAPR1, KLF3, SIX4, AP180, GRID2, PACN1, LASP1, RAI1, NOTC3, SALL3, SPTB2, ARI3A, NUP62, PHC1, TFE3, PAN3, TIF1A, SF01, SYN2, SBNO1, CRTC1, RIPR1, GIT1, PKP4, ABLM3, ARMX2, CE170, Q6AXD2, NIPBL, FBX41, RPRD2, WWC2, Q6P1J1, Q6P5E3, UGGG1, SPRE3, Q6P9N8, AHDC1, PTN23, TRAK1, DLGP3, NYAP1, DHX29, NFRKB, MAGI1, Q6XZL8, CNOT1, SYNE2, IF2A, PICAL, PLPR4, PLPR3, CCNT2, PRC2A, MAP6, MCAF1, RERE, NU214, SESD1, UBP2L, C2C2L, CNKR2, SLIK5, RHG32, LPP, NELFA, C42S2, TB10B, TGO1, RFOX3, SP130, ANS1B, ZC3HE, ZC21A, BAIP2, EMSY, KAT6B, RELL2, LIPA2, CNOT4, TOX4, GASP2, CREST, KDM4A, GRIN3, KAT6A, ZN609, PAK5, A16L1, SI1L1, SH3R3, SKA3, RBM14, Q8C5J0, CNOT2, WDR26, UBA6, ANK2, DIDO1, SYNPO, VCIP1, FHI2A, NUP88, NED4L, SET1B, TENS2, OGT1, NAV1, STAU2, AFG32, S4A8, ZBT20, HS12A, GLT18, UNC5A, AGFG1, FRRS1, KCNQ3, PO121, T2FB, MTSS1, Q8R2E1, NUP35, MAVS, SGIP1, HNRL1, PP16B, CCG8, SFPQ, UBAP2, NCOA5, AJUBA, DCP1A, TWF1, ALS2, ETFD, CIC, GRIP1, GORS2, NONO, ZN281, CT2NL, RN111, ANR17, PPP6, RBM7, CYGB, SARNP, DLGP1, SUN1, TM263, GON4L, PLIN3, MYPT1, NBEA, ZN704, RBP2, ARHG7, RTN3, NUDT3, TULP4, Q9JIZ5, PAR6G, SCAM5, PRG4, ZN207, SRCN1, ASAP1, DREB, ULK2, ADDA, PCLO, UBQL2, FBX6, PCM1, SYT7, CRY2, FOXO1, MAST1, LYPA2, TEN3, GANP, DEMA, E41L3, ZO2, BAG6, E41L1, RM40, GRIA3, S4R294, V9GWU7, V9GX40
Species: Mus musculus
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Zaro BW, Batt AR, Chuh KN, Navarro MX, Pratt MR. The Small Molecule 2-Azido-2-deoxy-glucose Is a Metabolic Chemical Reporter of O-GlcNAc Modifications in Mammalian Cells, Revealing an Unexpected Promiscuity of O-GlcNAc Transferase. ACS chemical biology 2017 12(3) 28135057
Abstract:
Glycans can be directly labeled using unnatural monosaccharide analogs, termed metabolic chemical reporters (MCRs). These compounds enable the secondary visualization and identification of glycoproteins by taking advantage of bioorthogonal reactions. Most widely used MCRs have azides or alkynes at the 2-N-acetyl position but are not selective for one class of glycoprotein over others. To address this limitation, we are exploring additional MCRs that have bioorthogonal functionality at other positions. Here, we report the characterization of 2-azido-2-deoxy-glucose (2AzGlc). We find that 2AzGlc selectively labels intracellular O-GlcNAc modifications, which further supports a somewhat unexpected, structural flexibility in this pathway. In contrast to the endogenous modification N-acetyl-glucosamine (GlcNAc), we find that 2AzGlc is not dynamically removed from protein substrates and that treatment with higher concentrations of per-acetylated 2AzGlc is toxic to cells. Finally, we demonstrate that this toxicity is an inherent property of the small-molecule, as removal of the 6-acetyl-group renders the corresponding reporter nontoxic but still results in protein labeling.
O-GlcNAc proteins:
A2A5R8, A2A6U3, A2AF81, A2AG39, A2AIW9, A2AJ72, A2AJI1, A2AKV2, A2AL12, A2AMW0, A2AUR3, LAS1L, TRM1L, A5A4Y9, A6PWC3, B0QZF8, B1AU76, UPP, B7ZC19, B7ZP47, B8JJC1, D3YWF6, D3YWK1, D3YWS3, D3YYP4, E9PX53, E9Q066, I2BP2, E9Q4Q2, E9Q5L7, E9Q7W0, E9QP59, F8WGW3, G3UX26, G3UYZ0, G3UZ44, G3X972, H3BKW0, H7BWX9, GTPB1, AIP, ATOX1, HDAC1, GSH0, DHX15, IKBE, AKAP2, SLK, IMPCT, IF6, ACOT1, NMT1, DHB12, SRPK1, ZN326, KLC1, RPP30, IDHC, CASP8, GCR, TYSY, RIR1, S10AA, LEG1, G3P, TPIS, PRDX3, CBX3, TISD, CATA, IMDH2, NFKB1, MAP4, CEBPB, CDK4, FKBP4, HMGB2, KAP3, MP2K1, RANG, PTN11, FBRL, PTN12, FMR1, HMGCL, DYN1, CAP1, STAT1, STAT3, PURA, ALD2, SIPA1, PURA2, GSHR, FOSL2, FOSL1, GSTM5, PCY1A, VATA, HDGF, UBP10, RHOX5, HMGA2, CCHL, NUB1, FAF1, ZNRD2, TB182, PCBP1, ARL1, PFD3, TCTP, HMGB1, DYL1, UB2L3, HDAC2, ELAV1, 4EBP2, PYRG1, TCPB, SPTC2, PSME2, BOP1, WBP2, XDH, HMMR, E2AK2, CO6A1, FABP5, LARP7, CNN2, PP4R2, RM10, Q3TFP0, GUAA, FUBP2, TRADD, CTU2, Q3U4W8, SNX27, BABA1, EDC4, COBL1, SKAP2, ARH40, CSTOS, LRRF1, ZMAT1, Q45VK5, JIP4, MDC1, Q5SUW3, SRC8, SAMH1, KHDR1, SPB6, CAPR1, PAPS1, TS101, PA1B2, FNTA, IGBP1, FSCN1, FXR1, CBX5, RAI1, MELK, FOXC2, DBNL, CYTB, NDRG1, RALY, GPDM, RAB3I, F120A, NOP58, Q6DFZ1, TPM4, Q6NXL1, Q6NZD2, TNPO3, SMHD1, UGGG1, UBXN7, TXLNA, DC1L2, KI18B, JUPI2, LARP1, CAND2, ACAP2, HNRPQ, SPAG7, ATX2L, MAP6, ELP1, PJA2, PGRC2, KCMF1, Q80VB6, FA98B, WDTC1, CPPED, LPP, PEF1, IF4B, ATG4B, FTO, Q8BH80, PRUN1, AHSA1, RCC2, NCEH1, LSS, FBLN3, PPR18, SRRM2, MSRB3, PPME1, RL1D1, TBCD4, NHLC2, MAP1S, TLK1, CND2, RAE1L, SEP10, ZFP57, UBA6, UBA3, STON1, PPM1F, GNL3, PUR1, HMCS1, Q8K0C7, PDXK, ANGE2, LRC41, SDE2, DNM1L, ANLN, MATR3, CBR3, MEPCE, ERF3A, DC1L1, SPART, TDIF2, HEXI1, SNP47, UBP15, MAVS, UBXN4, ACSF2, MICU1, ZNG1, BACH, ISOC1, IPYR2, CSDE1, PIP30, GCSH, Q91X76, DUS3L, BAG2, KCC1A, TTC1, HNRLL, RIN1, PP6R3, MARC2, DBR1, ATAD3, PSIP1, NXF1, NONO, PLST, RRAGC, VMA5A, TARA, DDAH2, TADA1, GRPE1, ABD12, NU155, OGFR, NPM3, GLOD4, COPRS, DPOE4, MIEN1, TRAP1, VATG1,