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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, HCFC1, 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, TLE3, 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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