panorama0000ProteomicsChi NguyenMus Musculushttps://panoramaweb.org/tObdcX.urlchi.nguyen@isas.deLeibniz-Institut für Analytische Wissenschaften – ISAS – e.V. GermanyPanoramaPublicSo far, mass spectrometry-based targeted proteomics is the most sensitive approach to answer and address specific biological questions in an accurate and quantitative fashion. However, the data analysis design used for such quantification varies in the field leading to discrepancies in the reported values. In this study, different quantification strategies based on calibration curves were evaluated and compared. The best accuracy and coefficient of variation was achieved by ratio to ratio calibration curves. We applied the ratio to ratio quantification approach to analyze very low abundant insulin signaling proteins such as PIK3RA (0.10-0.93 fmol/μg), AKT1 (0.1-0.39 fmol/μg), and the insulin receptor (0.22-2.62 fmol/μg) in a fat cell model and demonstrated the adaptation of this pathway at different states of insulin sensitivity.Analytical comparison of absolute quantification strategies to investigate the insulin signaling pathway in fat cells.Li Tingting T, Hentschel Andreas A, Ahrends Robert RSoluble, Insulin B Chain, dmBest1, PRKBA, Akt/PKB, dP60, PNT-P1, DAKT1/PKB, DmelCG17077, Addresses, Receptors, PI3K, insuline humaine, BcDNA:LD15217, Dp110, EY3-1, Pnt, sci, AKT1, Model Calibration, 11621, DmelCG6264, CD220, PI-3 kinase, p110-alpha, Techniques, anon-3B1.2, png, DmelCG2699, Method, human insulin, ARB, HOW, How, Analysis, phosphatidylinositol 3-kinase activity, D-ets-2, 3520, l(3)j5D5, IKKg, Insulin Receptor beta Subunit, KEY, Key, Pnt-P1, Mass Spectrum Analysis, 24B, insulin, me75, Dp60, Analyses, Pi3K92D, CG4141, developmental field, catalyst activity, stru, Pi3k, PI3-kinase activity, l(3)S053606, PI[[3]]K, CG10293, D17Mit170, PI3Kgamma, PKB|Akt, T1, RacPK, allergic reaction, MCAP, l(3)j5B5, insulin human, INSR, Methodological Studies, PI3K 68D, Pi3K, PI3k, MCMTC, ATP - 1-phosphatidyl-1D-myo-inositol 3-phosphotransferase activity, single organism signaling, insulinum humanum, 0904/17, PI3K68D, Regular, CG6264, dakt, P110DELTA, PtdIns-3-kinase activity, rea, D-Ets-2, ets94F, future organ, 0123/09, Procedure, l(3)89Bq, Tl3, Tl2, Spectrum Analysis, CG2699, Regular Insulin, dPIK, 1-phosphatidylinositol 3-kinase activity, Spectroscopy, DmelCG2650, insulin (recombinant), PKB/Akt, PKB/AKT, AKT/PKB, DmIKKgamma, dAkt, dAKT, SZ1, dIKK, type-1 PI3K, Kenny, p-Akt, DAkt, TU15B, Soluble Insulin, Sensor, dBest1, l(3)04226, CG11621, DPKB, Insulin Receptor alpha Chain, pntP2, Pointed-P1, pAkt, xAct, dbest1, Ets94F, IKK-gamma, Spectrometry, insulina humana, 0.9 gene, fmol, Methodological, PI3K 68_D, Methodological Study, dAKT/dPKB, PKB/dAKT, DmelCG4141, v-akt1, APDS, ptd, data analysis, PntP2, p110D, Dakt, Insulin Receptors, DmelCG16910, Sensor Calibration, PntP1, E(E2F)3D, PI3KBETA, dPKB, Sensor Calibrations, anon-EST:Liang-2.39, PNTP2, p120-PI3K, PNTP1, droPIK57, RAC-ALPHA, DRAC-PK85, 0998/12, DmelCG4006, Procedures, Peptidomics, P62, ETS2, PI3K92E, Ets2, number, ATAKT1, Gene, Insulin B, Spectrum Analyses, anon-WO0118547.380, type I phosphatidylinositol kinase activity, Insulin Receptor, presence, dIKK-gamma, EG:155E2.1, phosphatidylinositol 3-kinase, Dpkb, organ field, method, PIK3, Insulin Receptor Protein-Tyrosine Kinase, DMPOINT1A, pntegfr, 0608/07, v-akt, akt-1, Pi3Kp60, IR, sensitive, p60, method used in an experiment, Insulin, PI3K-dp110, DmIKK-gamma, Mass, Studies, Gene Products, dPI3K, field, Low, anon-92Ed, dmIKKgamma, IKK[[gamma]], sensitivity, Technique, Mass Spectroscopy, Dp110/PI3K, PI3K-68D/E, class I, Chain, study, l(3)s2612, pointed-RC, VMD2, F18A8.2, PI3'K, BMD, Pi3Kp110, Insulin Receptor Protein Tyrosine Kinase, EK3-2, P110BETA, Study, l(3)07825, Iletin, PI3K-68D, data processing, PI3K21B, Insulin recombinant, IKK, AKT, Akt, DmelCG10293, DRAC-PK, CG17077, Insulin-Dependent Tyrosine Protein Kinase, MCM, K+ transporter 1, Model, l(3)j1B7, RP50, Cpk, Insulin Receptor beta Chain, dP110, Ets, cou, akt, clone 2.39, p120, class II, type III phosphoinositide 3-kinase activity, Proteins, p110, Vps34p, DAkt1, DAKT1, qkr, l(3)S090417, PI(3)K, cpk, PI3K-92E/Dp110, IKKgamma, count in organism, PIK3C1, MS, Lr, ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase activity, PKB, Sodium Insulin, CWS6, vps34, CWS5, Model Calibrations, KH93F, Protein, Insulin A Chain, PKB-ALPHA, dp110, D-Akt, F18A8_2, CG4006, Sodium, Novolin, PI3CG, pnt-P1, pnt-P2, Mass Spectrum Analyses, l(3)s118306, who, PI-3-K, Dakt1, Mass Spectrum, Dbest, Exubera, pkb, best, PI3K-Dp110, akt1, dAkt1, Protein., Dmikkgamma, DRAC-PK66, class III, Who/How, DmelCG11621, p110gamma, CG16910, plan specification, Protein Gene Products, Gene Proteins, dAkt/PKB, Ets58AB, dakt1, CLOVE, signalling process, Data, concentration, Calibration, IMD14, PI3K_68D, PI3K-92D, PKBalpha, Calibrations, Bra, qkr[93F], Dmp110, dAKT1, RAC, Rac, Insulin Receptor alpha Subunit, BEST, 0.9, Data Analyses, POINT, POTASSIUM TRANSPORTER, CG8705insulinum humanum, Soluble, DmelCG3352, SCARB3, Insulin B Chain, Regular, BODYFAT, CT11259, l(2)gd-1, BcDNA:LD22582, R74677, signal transduction by conformational transition, GPIIIB, BcDNA.LD22582, insuline humaine, l(2)gd2, Insulin B, l(2)ft, fat, hFat1, BDPLT10, Dm Fat, CD36, ME5, signal transduction by trans-phosphorylation, Regular Insulin, insulin (recombinant), signaling cascade, Body Fat, Platelet glycoprotein IV, Sodium Insulin, CDHF7, PASIV, human insulin, Insulin, Insulin A Chain, PAS-4, Scarb3, Cell., Sodium, CG3352, Novolin, Cph1, PAS IV, signalling cascade, GP3B, Soluble Insulin, Cph-1, fat tissue, l(2)k07918, CDHR8, signal transduction by protein phosphorylation, Platelet collagen receptor, Chain, insulin, Exubera, Leukocyte differentiation antigen CD36, adipose system, insulina humana, CHDS7, l(2)gd-l, 79/18, CG1945, Fatty acid translocase, Iletin, signalling pathway, insulin human, l(2)fd, Insulin recombinant, signaling pathway, fatty depot, GP4, adipose, Faf, DmelCG1945, Ft, Glycoprotein IIIb, Thrombospondin receptor, l(2)fat, l(2)24Da, bodyfat, signal transduction by cis-phosphorylation, GPIV, Fat, FAT, CPH, l(2)79/18insulinum humanum, Soluble, DmelCG3352, SCARB3, Insulin B Chain, Regular, BODYFAT, CT11259, l(2)gd-1, BcDNA:LD22582, R74677, signal transduction by conformational transition, GPIIIB, BcDNA.LD22582, insuline humaine, l(2)gd2, Insulin B, l(2)ft, fat, hFat1, BDPLT10, Dm Fat, CD36, ME5, signal transduction by trans-phosphorylation, Regular Insulin, insulin (recombinant), signaling cascade, Body Fat, Platelet glycoprotein IV, Sodium Insulin, CDHF7, PASIV, human insulin, Insulin, Insulin A Chain, PAS-4, Scarb3, Cell., Sodium, CG3352, Novolin, Cph1, PAS IV, signalling cascade, GP3B, Soluble Insulin, Cph-1, fat tissue, l(2)k07918, CDHR8, signal transduction by protein phosphorylation, Platelet collagen receptor, Chain, insulin, Exubera, Leukocyte differentiation antigen CD36, adipose system, insulina humana, CHDS7, l(2)gd-l, 79/18, CG1945, Fatty acid translocase, Iletin, signalling pathway, insulin human, l(2)fd, Insulin recombinant, signaling pathway, fatty depot, GP4, adipose, Faf, DmelCG1945, Ft, Glycoprotein IIIb, Thrombospondin receptor, l(2)fat, l(2)24Da, bodyfat, signal transduction by cis-phosphorylation, GPIV, Fat, FAT, CPH, l(2)79/18Crbs, Soluble, DRAC-PK85, Insulin B Chain, DmelCG4006, dmBest1, PRKBA, Akt/PKB, Peptidomics, DAKT1/PKB, Addresses, number, Receptors, ATAKT1, Gene, insuline humaine, far, Insulin B, Spectrum Analyses, AKT1, Model Calibration, anon-WO0118547.380, Insulin Receptor, presence, DmelCG6264, DmelCG6383, CD220, Dpkb, organ field, Insulin Receptor Protein-Tyrosine Kinase, v-akt, akt-1, Crumbs, IR, sensitive, human insulin, Insulin, Mass, Gene Products, ARB, field, Low, Analysis, sensitivity, Mass Spectroscopy, Insulin Receptor beta Subunit, Mass Spectrum Analysis, Chain, study, CT19912, insulin, 0509/20, me75, Analyses, VMD2, F18A8.2, developmental field, 1384/04, crumb, BMD, Insulin Receptor Protein Tyrosine Kinase, D17Mit170, PKB|Akt, T1, RacPK, allergic reaction, Iletin, insulin human, INSR, data processing, Insulin recombinant, AKT, Akt, Lipocytes, DRAC-PK, Lipocyte, Sensitivity, Insulin-Dependent Tyrosine Protein Kinase, lipocyte, K+ transporter 1, Model, l(3)j1B5, single organism signaling, RP50, Insulin Receptor beta Chain, insulinum humanum, Regular, CG6264, cou, dakt, CG6383, akt, Proteins, DAkt1, future organ, DAKT1, l(3)89Bq, Tl3, Tl2, Spectrum Analysis, Cell, Fat Cells, l(3)07207, Regular Insulin, Spectroscopy, count in organism, insulin (recombinant), PKB/Akt, PKB/AKT, AKT/PKB, adipose cell, MS, Lr, PKB, Sodium Insulin, dAkt, dAKT, CWS6, Model Calibrations, Protein, Insulin A Chain, PKB-ALPHA, CRB, Crb, D-Akt, p-Akt, F18A8_2, CG4006, Sodium, sensitive., Novolin, adipocyte, l(3)S058104, DAkt, TU15B, Soluble Insulin, Mass Spectrum Analyses, Sensor, dBest1, l(3)04226, Dakt1, Adipocyte, Mass Spectrum, DPKB, Insulin Receptor alpha Chain, Dbest, Exubera, pkb, pAkt, best, akt1, dAkt1, xAct, DRAC-PK66, dbest1, Spectrometry, insulina humana, Specificity, fmol, dAKT/dPKB, PKB/dAKT, v-akt1, fat cell, Protein Gene Products, Gene Proteins, data analysis, dAkt/PKB, dakt1, Dakt, Insulin Receptors, signalling process, Specificity and Sensitivity, Data, Fat Cell, Calibration, Sensor Calibration, dPKB, PKBalpha, Cells, Calibrations, Bra, Sensor Calibrations, dAKT1, RAC, Rac, Fat, Insulin Receptor alpha Subunit, l(3)S050920, BEST, Data Analyses, POTASSIUM TRANSPORTER, RAC-ALPHA0falseAnalytical Comparison of absolute quantification strategies to investigate the Insulin signaling pathway in fat cellsMass spectrometry based targeted proteomics is the most sensitive approach to answer and address specific biological questions in an accurate and quantitative fashion. However, the data analysis design used for such quantification varies in the field leading to discrepancies in the reported values. In this study, different quantification strategies based on calibration curves were evaluated and compared. The best linearity was achieved when creating a calibration curve using the intensity to amount method. However, the best accuracy and coefficient of variation was achieved by ratio to ratio calibration curves. In addition, we also demonstrated how a LLOQ estimation in combination with one-point calibration can be utilized to conduct a less laborious strategy for absolute quantification. Finally, we applied the most accurate method ratio to ratio quantification to analyze very low abundant insulin signaling proteins such as PIK3 (0.34-0.9 fmol/mg), AKT1 (0.33-0.54 fmol/mg) and the Insulin receptor (0.34 -3.98 fmol/mg) and demonstrated that upon pathway perturbation the concentration of key proteins is dynamically adapted.Mon Jun 13 00:00:00 GMT+01:00 2022PXD0262041009034964541