Progenesis LC-MS and Phenyx Une combinaison unique d un logiciel d analyse différentielle en LC-MS et d une plateforme d identification et de caractérisation de protéines et peptides à partir de données MS/MS. Identifiez les vraies différences en Protéomique
Nonlinear Dynamics Ltd. Who are we? Nonlinear Dynamics is dedicated to solving your problems in the fields of Proteomics, Genomics and Biomarker Discovery. This is achieved by applying our core strength in developing innovative, high quality data analysis solutions What is Progenesis LC-MS? Progenesis LC-MS is an advanced proteomics research solution for the label-free quantitative analysis of complex samples It uses unique technology to deliver the advantages of speed, objectivity and statistical power It applies simple workflows so you can easily visualise complex analysis data MS-MS protein identifications can be integrated back into the software and linked with the original analysis results
Nonlinear Dynamics Ltd. Qui sommes nous? La mission de Nonlinear Dynamics est d essayer de résoudre les problèmes en Protéomique, Génomique et la découverte de Biomarqueurs. Notre force est de pouvoir développer des solutions d analyses innovatrices, de grande qualité et simple d utilisation Progenesis LC-MS? Progenesis LC-MS est un outil de recherche protéomique qui permet l analyse quantitative d échantillons complexes sans mass tags Il utilise une technologie unique alliant vitesse, objectivité et puissance statistique. Vous êtes guidé au travers du logiciel, vous permettant ainsi de visualiser facilement des données complexes. L identification des protéines grâce aux spectres MS-MS peut être liée aux résultats de la quantification à postériori.
A workflow for the differential analysis of complex samples 4
A workflow for the differential analysis of complex samples Obtain sequence data using targeted LC-MS/MS Generate and export MS-MS search list 5
Progenesis LC-MS Workflow : Data Modelling 6
Progenesis LC-MS Workflow : Alignment Total Ion Chromatogram LC-MS run Alignment Automatic Uses mass and retention time data Section prior to alignment Section after alignment Example of pre- and post- alignment on LC-MS run 7
Progenesis LC-MS Workflow : Feature Detection Intelligent detection of co-eluting peptides 8
Overlapping Feature Detection Feature detection in Progenesis LC-MS enables the correction detection and quantitation of overlapping pepties (features) 9
Viewing MS2 data for overlapping features In Protein ID you can see that the 2 overlapping features only feature 7959 actually has MS2 spectra available whereas feature 9896 does not. Furthermore according to the Stats in the results view 7959 has a high p value and is therefore unlikely to be of significance (see previous slide) 10
Significant feature masked by insignificant feature Examining Feature 9896 in the 1D views shows a that it is clearly differentially expressed across the groups with consistent behaviour within each group 11
Focusing on Data that has Protein Id information from Phenyx Following data analysis in the LC-MS Workflow. The detected features with MS2 spectra can be searched using Protein search engines such a Phenyx, Mascot and Sequest. This data can be used with the Multivariate Statistical analysis tools to focus the data exploration by using only identified features. 12
Correlation analysis of identified features (up regulation) Experiment looking at changes between two conditions focusing on Identified features with P<0.05 (tagged selection for up regulation) 13
Correlation analysis of identified features (up regulation) Experiment looking at changes between two conditions focusing on Identified features with P<0.05 (tagged selection for down regulation) 14
Examine Protein ID table showing modifications Protein ID table shows Peptide Ids and highlighted modifications (in red) for tagged group of features from correlation Analysis (Previous slide) 15
Export Data from LCMS to Excel Export table and sort out put in Excel to show features related to same protein Peak number m/z Retention time (s) Charge Max fold Protein Ids change Anova 1 2 3 4 5 6Protein Ids MS2 No IDs p<0.05 Increased with ID_p<0.05 Decreased with ID_p0.05 Score Protein Sequence 6004 766.863 4714.22 2 1.347883 0.183617 2194.685 3175.85 3885.161 2393.035 2301.66 2172.144x 50.561433T_MOUSE AVTEQGAELSNEER 1 5631 687.8814 6921.8 2 1.281072 0.395884 2310.101 0 1131.073 1027.328 1777.355 1603.709x 26.371433T_MOUSE MEKTELIQKAK 1 3970 320.5342 4836.18 3 1.8438261 0.963989 87.985 2316.648 685.4413 464.4068 750.8145 460.6821x 22.27ACINU_MOUSE SGQKSALVK 2 2913 488.7279 4515.36 2 1.2660749 0.733886 2554.292 8568.832 4710.441 3756.794 5336.329 3412.904x 76.89ACTA_MOUSE AGFAGDDAPR 3 4367 599.765 4240.17 2 1.8221227 0.178853 7010.205 10382.06 18993.61 6065.757 8844.706 5058.486x 59.73ACTA_MOUSE DSYVGDEAQSK 3 4289 677.8153 4064.52 2 2.2330606 0.065808 2586.745 3864.716 5572.441 2546.711 1135.017 1702.766x 57.13ACTA_MOUSE DSYVGDEAQSKR 3 2777 581.313 5707.3 2 3.0287321 0.04079 11602.35 14616.99 6883.063 5101.086 4029.897 1798.476x x 37.87ACTA_MOUSE EITALAPSTMK 3 4630 586.2891 4429.34 2 3.7918972 0.056868 1465.166 5050.623 3310.963 1379.281 419.8772 792.3549x 22.5ACTB_MOUSE HQGVMVGMGQK 4 5920 758.855 4910.09 2 5.1515068 0.049991 716.9212 3981.502 1530.015 500.0198 360.4209 348.6109x x 23.8ACTB_MOUSE QEYDESGPSIVHR 4 3925 506.2391 4904.92 3 1.2722637 0.794963 1579.104 5305.681 5228.736 2702.463 3780.687 3038.085x 29.9ACTB_MOUSE QEYDESGPSIVHR 4 5680 895.949 7283.75 2 1.2076866 0.64136 10724.64 2590.458 5125.407 1461.603 3121.92 10685.76x 63.94ACTB_MOUSE SYELPDGQVITIGNER 4 5584 448.241 5660.35 3 1.0780476 0.743535 568.7565 1065.383 492.6752 609.9109 637.1854 1045.711x 29.48ALDOA_MOUSE ADDGRPFPQVIK 5 4700 470.7458 4367.96 2 2.285003 0.340829 964.5077 3920.542 3456.35 1660.714 0 1989.784x 50.36ALDOA_MOUSE ELSDIAHR 5 3013 666.8538 5579.31 2 1.0531138 0.678945 7790.474 5206.824 4245.773 6199.731 6442.482 5516.704x 88.42ALDOA_MOUSE GILAADESTGSIAK 5 5591 601.6417 4087.01 3 1.73573 0.039788 1822.412 2789.269 3221.866 5090.587 4672.46 3833.877x x 32.32ALDOA_MOUSE RLQSIGTENTEENRR 5 Same Protein 4547 966.5789 8908.83 2 6.2852951 0.007587 14034.78 17059.14 43486.03 138924.1 118773.8 211059.1x x 22.23ARI4B_MOUSE ADKIVRPADKNVPKIK 6 6940 997.0437 8481.4 2 1.9909338 0.293029 2796.576 4005.805 10785.36 1662.336 2808.581 4363.001x 20.44ARI4B_MOUSE KDTTDKALKPQTKRGK 6 6708 502.3143 1066.11 4 107.88138 0.003984 531.9819 430.1969 2544.397 65118.77 269783.5 43391.91x x 20.61ARI4B_MOUSE KQKRSHKATVVNNKKK 6 6125 925.4335 6332.14 2 3.0465809 0.137311 97.05531 1103.987 565.296 2121.13 942.1662 2317.995x 69.94ASF1A_MOUSE VGYYVNNEYTETELR 7 4205 650.8523 5506.16 2 1.397399 0.254623 4174.565 4282.518 3312.138 4163.824 1484.564 2773.845x 44.54BXDC1_MOUSE GGNANATVTQVLR 8 5821 722.3469 6426.45 2 2.8131621 0.007527 2806.913 1850.748 1667.556 782.476 890.5624 575.3983x x x 85.59BXDC1_MOUSE NISQDTFGTTFGR 8 6704 763.4401 7005.49 2 3.7462334 0.058137 3068.596 1806.989 1326.645 170.935 1006.253 478.4029x 65.27BXDC2_MOUSE FLVQNIHTLAELK 9 6099 549.9777 5395.32 3 1.3400627 0.169389 1226.029 1568.194 1484.828 1075.444 751.3326 1366.396x 22.23CBX3_MOUSE SKKKRDAADKPR 10 6108 497.2562 6011.39 3 5.4317503 0.012459 557.8398 396.7459 224.0456 42.28826 59.40319 115.2978x x 46.84CBX3_MOUSE WKDSDEADLVLAK 10 5457 745.3802 6011.39 2 4.4828783 0.005397 1863.82 971.7652 838.0919 308.2925 287.0654 224.1328x x 58.5CBX3_MOUSE WKDSDEADLVLAK 10 16
Export Data from LCMS to Excel Order table on Tags and examine Protein IDs for increased expression Suz + MS Suz - MS Tags Best peptide match Peak number m/z Retention time (s) Charge Increased Decreased Max fold Protein Ids with with change Anova 1 2 3 4 5 6Protein Ids MS2 No IDs p<0.05 ID_p<0.05 ID_p0.05 Score Protein Sequence 6004 766.863 4714.22 2 1.347883 0.183617 2194.685 3175.85 3885.161 2393.035 2301.66 2172.144x x x 50.561433T_MOUSE AVTEQGAELSNEER 1 44 406.2004 3551.34 2 2.1047441 0.007126 505461.5 664676.8 651597.2 1546759 1035561 1251967x x x 54.35H12_MOUSE GTGASGSFK 2 5275 429.7376 606.476 2Infinity 1.25E-06 0 0 0 68093.06 38035.31 30085.63x x x 30.31H13_MOUSE AASGEAKPK 3 5140 329.5254 939.146 3Infinity 5.57E-06 0 0 0 12047.53 6026.094 4424.829x x x 27.62H13_MOUSE KAASGEAKPK 3 5126 329.5255 1066.11 3Infinity 4.07E-06 0 0 0 21937.69 10657.19 8314.643x x x 31.83H13_MOUSE KAASGEAKPK 3 3687 422.7479 1679.34 2Infinity 1.12E-07 0 0 0 19169.66 18515.65 13094.41x x x 23.74H13_MOUSE KATGAATPK 3 4965 422.7479 1371.12 2Infinity 1.96E-09 0 0 0 6036.538 6320.057 5517.38x x x 26.52H13_MOUSE KATGAATPK 3 3918 422.7479 1601.11 2Infinity 1.01E-07 0 0 0 30734.35 29210.17 20709.73x x x 26.91H13_MOUSE KATGAATPK 3 4257 422.7479 1545.44 2Infinity 1.33E-07 0 0 0 17637.92 15886.53 11609.51x x x 29.31H13_MOUSE KATGAATPK 3 3791 422.7479 1619.84 2Infinity 1.17E-07 0 0 0 8511.676 8706.491 6120.808x x x 30.75H13_MOUSE KATGAATPK 3 2563 422.7478 2039.88 2Infinity 1.40E-07 0 0 0 14908.13 10292.35 10249.65x x x 36H13_MOUSE KATGAATPK 3 2637 422.7478 2061.58 2Infinity 8.86E-06 0 0 0 10615.21 6167.872 20648.73x x x 40.95H13_MOUSE KATGAATPK 3 2579 422.7478 2013.18 2Infinity 5.64E-08 0 0 0 12560.79 10742.23 8944.121x x x 45.75H13_MOUSE KATGAATPK 3 2361 422.7478 1990.88 2Infinity 2.37E-07 0 0 0 104458.8 85556.98 58691.79x x x 50.05H13_MOUSE KATGAATPK 3 1720 366.2213 650.576 4Infinity 1.37E-07 0 0 0 655666.7 947289.8 521515.5x x x 20.34H14_MOUSE AAKPKKTAAKKK 4 18 650.8461 6316.49 2 2.0526804 0.002983 1471542 1459416 1401671 3624100 2535672 2733730x x x 84.63H2A1F_MOUSE NDEELNKLLGR 5 2077 427.2117 2346.49 2Infinity 3.05E-07 0 0 0 35399.98 23367.95 20648.6x x x 20.07H2A3_MOUSE KGNYSER 6 1581 427.2116 2628.61 2Infinity 1.09E-07 0 0 0 68527.76 46885.58 44897.54x x x 20.91H2A3_MOUSE KGNYSER 6 2408 427.2115 2141.07 2Infinity 2.59E-06 0 0 0 18645.48 14602.64 7777.122x x x 28.55H2A3_MOUSE KGNYSER 6 4791 427.2116 1915.74 2Infinity 5.54E-07 0 0 0 39977.12 25281.21 21074.68x x x 32.89H2A3_MOUSE KGNYSER 6 2110 427.2117 2440.37 2Infinity 1.87E-08 0 0 0 82774.86 70755.25 60878.67x x x 33.35H2A3_MOUSE KGNYSER 6 1928 427.2116 2287.18 2Infinity 1.68E-06 0 0 0 84746.94 73742.06 35540.89x x x 35.18H2A3_MOUSE KGNYSER 6 2052 427.2117 2357.75 2Infinity 1.78E-07 0 0 0 38514.34 29319.71 23377.7x x x 36.26H2A3_MOUSE KGNYSER 6 2148 427.2117 2369.02 2Infinity 9.48E-08 0 0 0 46119.34 36824.7 29837.31x x x 37.51H2A3_MOUSE KGNYSER 6 2011 427.2117 2474.15 2Infinity 8.88E-08 0 0 0 35165.01 28193 23148.14x x x 38.57H2A3_MOUSE KGNYSER 6 2112 427.2117 2422.15 2Infinity 8.54E-08 0 0 0 41781.38 35127.84 27469.18x x x 38.74H2A3_MOUSE KGNYSER 6 1865 427.2117 2518.1 2Infinity 1.13E-08 0 0 0 144226.4 123445.5 108661.4x x x 39.35H2A3_MOUSE KGNYSER 6 1754 427.2117 2559.83 2Infinity 2.13E-08 0 0 0 97966.99 79228.55 71505.73x x x 40.7H2A3_MOUSE KGNYSER 6 1922 427.2115 2211.55 2Infinity 3.07E-07 0 0 0 56112.51 31832.26 49310.16x x x 40.77H2A3_MOUSE KGNYSER 6 1673 427.2115 2243.3 2Infinity 3.40E-05 0 0 0 162060.2 22311.21 73466.47x x x 40.78H2A3_MOUSE KGNYSER 6 1562 427.2116 2621.61 2Infinity 3.19E-08 0 0 0 118016.9 89293.38 84292.3x x x 40.8H2A3_MOUSE KGNYSER 6 1703 427.2115 2253.14 2Infinity 2.99E-06 0 0 0 140228.5 65648.52 48177.92x x x 40.8H2A3_MOUSE KGNYSER 6 2019 427.2117 2481.18 2Infinity 7.39E-08 0 0 0 41270.04 30998.63 27906.04x x x 40.9H2A3_MOUSE KGNYSER 6 17
Progenesis LC-MS Workflow : Multivariate Statistics 18
Progenesis LC-MS Workflow : MS-MS Data Integration Integration of Protein IDs 19
Phenyx Overview 20
Phenyx 2.5 Get more highly Confident Identifications Powerful and flexible scoring system Enriched search parameters (2-rounds, submission profiles, ) Extensive use of annotations in databases (PTMs, variants, AA modifs in Swiss-Prot, TrEMBL...) Enhanced accuracy and reliability thanks to the manual validation functionality and the unique conflict resolution algorithm 21
Phenyx 2.5 Plus de confiance dans les identifications Un score très puissant Des paramètres de recherche sophistiqués (2 tours; sauvegarde des paramètres) Usage total des annotations présentes dans les bases de données(ptms, variants, AA modifs in Swiss-Prot, TrEMBL...) Précision et confidence accrue grâce à la fonction de validation manuelle et l algorithme de résolution de conflit 22
Phenyx 2.5- Powerful and Interactive Platform for Data Management Full Visualization (interactive Web interface, dynamic views for, proteins overview and details, spectra overview and peptides details ) Comparison/cross-validation of Phenyx, Mascot, Sequest and X!Tandem results via the results comparison tool Expanded Exports/Reports Functionalities (Global Excel report, XML/PDF/text exports, FDR, calibration status of the instrument ) One-click results sharing with 3 levels of rights (read only, resubmission, deletion ) Automated data submission with the Phenyx Daemon Easy integration with your own Proteomics pipeline and third-party applications (Scaffold, TPP ) and bioinformatics pipelines (thanks to Web services, API, battery of Perl scripts, Perl modules, XML result files, command line interface ) 23
Phenyx 2.5- Plateforme puissante et interactive Nombreux outils visuels(interface Web interactif, vues dynamiques générales et en détail des protéines, spectres et peptides ) Comparaison et validation croisée des résultats de Phenyx, Mascot, Sequest et X!Tandem via l outil de comparaison Fonctions d export et de rapports étendus (rapport Excel global, exports XML/PDF/text, FDR, rapport de calibration de l instrument) Echange des résultats One-click avec 3 niveaux de droits d accès (read only, resubmission, deletion ) Soumission automatisée grâce à Phenyx Daemon Intégration simple dans votre flux Protéomique, dans les applications connexes (Scaffold, TPP ) et votre bioinformatique (grâce aux services Web, API, batterie de scripts Perl, modules Perl, fichiers de résultats XML, interface ligne de commande ) 24
To try Phenyx, a public server is available at: http://phenyx.vital-it.ch/pwi More info on Phenyx at: www.phenyx-ms.com
Progenesis LC-MS and Phenyx A unique LC-MS expression analysis tool combined with a software platform dedicated to the identification and characterization of proteins and peptides from MS/MS data. Identify the real differences in your proteomics data
Progenesis LC-MS and Phenyx Une unique combinaison d un logiciel d analyse différentielle en LC-MS et d une plateforme d identification et de caractérisation de protéines et de peptides à partir de données MS/MS. Identifiez les vraies différences en Protéomique www.nonlinear.com
Progenesis LC-MS and Phenyx A unique LC-MS expression analysis tool combined with a software platform dedicated to the identification and characterization of proteins and peptides from MS/MS data. Identify the real differences in your proteomics data