FDR calculation

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1

Hi,

I am doing 4 plex iTRAQ qauntative proteomics using OPENMS in KNIME, after creation of complete workflow based on TUTORIAL avialable in OPENMS site and the workflow are succesful. i applied FDR at level of 0.01 at both prot and pep level, i got following result. In that q value are all above 0.01. I donot know how to interpret this result? what is this q value? if q value is 0.98 or 0.87 or 0.5 , what is the meaning of this value?

PEPTIDE MZ SCORE RANK SEQUCENS PEPTIDE CHARGE aa before aa after score type  search identifier acessions 
525.2706299 0.879604672 0 (iTRAQ4plex)VPAPC(Carbamidomethyl)QYA 2 R H q-value XTandem_2016-07-02T07:27:58 sp|A6N7Y9|PIWL1_CHICK
499.3265076 0.93289225 0 (iTRAQ4plex)RVPSAAPR 2 S Y q-value XTandem_2016-07-02T18:34:24 sp|A7M7B9|NTH_CHICK
590.8082886 0.974264706 0 (iTRAQ4plex)T(Phospho)C(Carbamidomethyl)LPVNPR 2 Q C q-value XTandem_2016-07-02T05:20:51 sp|A7M7B9|NTH_CHICK
555.8346558 0.904509284 0 (iTRAQ4plex)HAQVKVER 2 I E q-value XTandem_2016-07-02T06:23:44 sp|A7XYH5|SOBP_CHICK
428.2645264 0.731034483 0 (iTRAQ4plex)GSPEPGPA 2 R G q-value XTandem_2016-07-02T11:41:38 sp|D0PRN2|NRX1B_CHICK
850.4468994 0.879604672 0 (iTRAQ4plex)FLPY(Phospho)DSREHRR 2 T D q-value XTandem_2016-07-02T07:27:58 sp|D3KCC4|CRNS1_CHICK
739.0878906 0.889967638 0 (iTRAQ4plex)SAAPRLPMALRIC(Carbamidomethyl)TVVC(Carbamidomethyl)R 3 R S q-value XTandem_2016-07-02T08:36:10 sp|D3KCC4|CRNS1_CHICK
753.7973633 0.908063301 0 (iTRAQ4plex)LPS(Phospho)DT(Phospho)ASRLSR 2 Q G q-value XTandem_2016-07-02T07:27:58 sp|E1C065|CEP41_CHICK
623.895752 0.731034483 0 (iTRAQ4plex)RQHLLS(Phospho)LR 2 P E q-value XTandem_2016-07-02T11:41:38 sp|E1C6Q1|PCH2_CHICK
461.2804871 0.974264706 0 (Glu->pyro-Glu)EDIPGPPA 2 K G q-value XTandem_2016-07-02T05:20:51 sp|E1C8P7|DSCL1_CHICK
461.2804871 0.974264706 0 (Glu->pyro-Glu)EDIPGPPA 2 K G q-value XTandem_2016-07-02T05:20:51 sp|E1C8P7|DSCL1_CHICK
603.8598633 0.974264706 0 (iTRAQ4plex)HLTLDPAAKP 2 R P q-value XTandem_2016-07-02T05:20:51 sp|E1C8P7|DSCL1_CHICK
683.4590454 0.858998145 0 (iTRAQ4plex)QVPPLGIKLTR 2 Q G q-value XTandem_2016-07-02T09:46:27 sp|F1N8V3|PHAR4_CHICK
683.4590454 0.858998145 0 (iTRAQ4plex)QVPPLGIKLTR 2 Q G q-value XTandem_2016-07-02T09:46:27 sp|F1N8V3|PHAR4_CHICK
475.8217773 0.974264706 0 (iTRAQ4plex)NVILQYG 2 K F q-value XTandem_2016-07-02T05:20:51 sp|F1N9S8|CARME_CHICK
748.4034424 0.93289225 0 (Glu->pyro-Glu)EGFIGLKDYATR 2 N V q-value XTandem_2016-07-02T18:34:24 sp|F1ND48|FBH1_CHICK
636.8753052 0.974264706 0 (iTRAQ4plex)LTTGDC(Carbamidomethyl)S(Phospho)T(Phospho)AAST(Phospho)PSR 3 G K q-value XTandem_2016-07-02T05:20:51 sp|F1NSM7|OC116_CHICK
451.7488403 0.873076923 0 (Acetyl)M(Oxidation)ALPRL 2 [ T q-value XTandem_2016-07-04T20:53:47 sp|F1NTD6|ASCC3_CHICK
470.3044128 0.838709677 0 (iTRAQ4plex)IPPVTLR 2 G W q-value XTandem_2016-07-02T09:46:27 sp|F1NY98|DSCAM_CHICK
447.6030579 0.863261944 0 (iTRAQ4plex)DS(Phospho)NGT(Phospho)P 2 K S q-value XTandem_2016-07-02T09:46:27 sp|F1NY98|DSCAM_CHICK
516.2779541 0.904509284 0 (iTRAQ4plex)S(Phospho)PIHTPR 2 I R q-value XTandem_2016-07-02T06:23:44 sp|O12940|TOM1_CHICK
520.9492188 0.889967638 0 (iTRAQ4plex)LSSQLAGM(Oxidation)T(Phospho)LGSR 3 N S q-value XTandem_2016-07-02T08:36:10 sp|O12940|TOM1_CHICK
639.8589478 0.904509284 0 (iTRAQ4plex)LS(Phospho)VIPEAGLR 2 W I q-value XTandem_2016-07-02T06:23:44 sp|O42273|TENP_CHICK
639.8589478 0.904509284 0 (iTRAQ4plex)LS(Phospho)VIPEAGLR 2 W I q-value XTandem_2016-07-02T06:23:44 sp|O42273|TENP_CHICK
639.8589478 0.904509284 0 (iTRAQ4plex)LS(Phospho)VIPEAGLR 2 W I q-value XTandem_2016-07-02T06:23:44 sp|O42273|TENP_CHICK
531.3022461 0.838709677 0 (Glu->pyro-Glu)EGVHLPHM(Oxidation) 2 R S q-value XTandem_2016-07-02T09:46:27 sp|O42273|TENP_CHICK
525.3007202 0.731034483 0 (iTRAQ4plex)YGAYFER 2 A I q-value XTandem_2016-07-02T11:41:38 sp|O42412|IOD3_CHICK
924.543457 0.789699571 0 (iTRAQ4plex)DPEAVYTTTGGKIPVR 2 D W q-value XTandem_2016-07-02T09:46:27 sp|O42422|EPHA7_CHICK
713.9199829 0.861538462 0 (iTRAQ4plex)STTPPSPLKAKR 2 A I q-value XTandem_2016-07-02T11:41:38 sp|O57415|RREB1_CHICK
568.6526489 0.905767669 0 (iTRAQ4plex)T(Phospho)C(Carbamidomethyl)QS(Phospho)SHL 2 K D q-value XTandem_2016-07-02T08:36:10 sp|O57598|ATOH7_CHICK
615.338623 0.853915663 0 (iTRAQ4plex)VNNHSGIVVM(Oxidation) 2 K Q q-value XTandem_2016-07-02T09:46:27 sp|O73775-1|FBLN1_CHICK
542.7857666 0.861538462 0 (iTRAQ4plex)RRMDPEH 2 R E q-value XTandem_2016-07-02T11:41:38 sp|O73790|SPB10_CHICK
336.202301 0.864864865 0 (iTRAQ4plex)ADPAALM(Oxidation)AS 3 K I q-value XTandem_2016-07-04T20:53:47 sp|O93344|AL1A2_CHICK
336.202301 0.864864865 0 (iTRAQ4plex)ADPAALM(Oxidation)AS 3 K I q-value XTandem_2016-07-04T20:53:47 sp|O93344|AL1A2_CHICK
336.202301 0.864864865 0 (iTRAQ4plex)ADPAALM(Oxidation)AS 3 K I q-value XTandem_2016-07-04T20:53:47 sp|O93344|AL1A2_CHICK
336.202301 0.864864865 0 (iTRAQ4plex)ADPAALM(Oxidation)AS 3 K I q-value XTandem_2016-07-04T20:53:47 sp|O93344|AL1A2_CHICK
813.1367798 0.93289225 0 (iTRAQ4plex)VPAHRPIPGAVPPPVPSAIPAVPG 3 R I q-value XTandem_2016-07-02T18:34:24 sp|O93367|TLX3_CHICK
813.1367798 0.93289225 0 (iTRAQ4plex)VPAHRPIPGAVPPPVPSAIPAVPG 3 R I q-value XTandem_2016-07-02T18:34:24 sp|O93367|TLX3_CHICK
651.8408203 0.731034483 0 (iTRAQ4plex)VPAPNMSTGER 2 D E q-value XTandem_2016-07-02T11:41:38 sp|P00368|DHE3_CHICK
651.8408203 0.731034483 0 (iTRAQ4plex)VPAPNMSTGER 2 D E q-value XTandem_2016-07-02T11:41:38 sp|P00368|DHE3_CHICK
618.8479614 0.974264706 0 (iTRAQ4plex)S(Phospho)RLLLSAPR 2 Q R q-value XTandem_2016-07-02T05:20:51 sp|P00508|AATM_CHICK
618.8479614 0.974264706 0 (iTRAQ4plex)S(Phospho)RLLLSAPR 2 Q R q-value XTandem_2016-07-02T05:20:51 sp|P00508|AATM_CHICK
878.5185547 0.974264706 0 (iTRAQ4plex)LGT(Phospho)GLRGGVHVKLPK 2 N I q-value XTandem_2016-07-02T05:20:51 sp|P00565|KCRM_CHICK
389.2703857 0.904509284 0 (iTRAQ4plex)KDGSAR 2 D I q-value XTandem_2016-07-02T06:23:44 sp|P00789|CANX_CHICK
921.5137939 0.974264706 0 (iTRAQ4plex)S(Phospho)VVFPYPLSERAPR 2 P A q-value XTandem_2016-07-02T05:20:51 sp|P01109|MYC_CHICK
434.9520569 0 0 (iTRAQ4plex)VLSAADKNNVK 3 M G q-value XTandem_2016-07-02T08:36:10 sp|P01994|HBA_CHICK
434.9520569 0 0 (iTRAQ4plex)VLSAADKNNVK 3 M G q-value XTandem_2016-07-02T08:36:10 sp|P01994|HBA_CHICK
434.9520569 0.889967638 0 (iTRAQ4plex)VLSAADKNNVK 3 M G q-value XTandem_2016-07-02T08:36:10 sp|P01994|HBA_CHICK
597.3429565 0.889967638 0 (Glu->pyro-Glu)EYGAETLER 2 E M q-value XTandem_2016-07-02T08:36:10 sp|P01994|HBA_CHICK
468.7975159 0.984550562 0 (iTRAQ4plex)LSDLHAH 2 K K q-value XTandem_2016-07-02T10:40:59 sp|P01994|HBA_CHICK
468.7975159 0.984550562 0 (iTRAQ4plex)LSDLHAH 2 K K q-value XTandem_2016-07-02T10:40:59 sp|P01994|HBA_CHICK
468.7975159 0.984550562 0 (iTRAQ4plex)LSDLHAH 2 K K q-value XTandem_2016-07-02T10:40:59 sp|P01994|HBA_CHICK
488.9335938 0.731034483 0 (iTRAQ4plex)TYFPHFDLSHG 3 K S q-value XTandem_2016-07-02T11:41:38 sp|P01994|HBA_CHICK
488.9335938 0 0 (iTRAQ4plex)TYFPHFDLSHG 3 K S q-value XTandem_2016-07-02T11:41:38 sp|P01994|HBA_CHICK
611.017395 0.889967638 0 (iTRAQ4plex)GM(Oxidation)VVGIKLDAGTAPLAGT 3 K N q-value XTandem_2016-07-02T08:36:10 sp|P07341|ALDOB_CHICK
440.2769775 0.879604672 0 (iTRAQ4plex)RHPGLR 2 P V q-value XTandem_2016-07-02T07:27:58 sp|P07850|SUOX_CHICK
440.2769775 0.879604672 0 (iTRAQ4plex)RHPGLR 2 P V q-value XTandem_2016-07-02T07:27:58 sp|P07850|SUOX_CHICK
452.2509766 0.864864865 0 (iTRAQ4plex)FAGDPPR 2 P H q-value XTandem_2016-07-04T20:53:47 sp|P07850|SUOX_CHICK
452.2509766 0.864864865 0 (iTRAQ4plex)FAGDPPR 2 P H q-value XTandem_2016-07-04T20:53:47 sp|P07850|SUOX_CHICK
452.2509766 0.864864865 0 (iTRAQ4plex)FAGDPPR 2 P H q-value XTandem_2016-07-04T20:53:47 sp|P07850|SUOX_CHICK
452.2509766 0.864864865 0 (iTRAQ4plex)FAGDPPR 2 P H q-value XTandem_2016-07-04T20:53:47 sp|P07850|SUOX_CHICK
506.9491882 0.838709677 0 (iTRAQ4plex)RLVEYLLDPTR 3 E Y q-value XTandem_2016-07-02T09:46:27 sp|P09484|ACHB2_CHICK
485.2944031 0.879604672 0 (iTRAQ4plex)TRPQPAR 2 D Y q-value XTandem_2016-07-02T07:27:58 sp|P0C7A1|ENASE_CHICK
430.9569397 0.636363636 0 (iTRAQ4plex)KVFVREAAAR 3 I A q-value XTandem_2016-07-02T09:46:27 sp|P0DJH7|CENPX_CHICK
421.9342041 0 0 (iTRAQ4plex)T(Phospho)VPIVNAAVR 3 V E q-value XTandem_2016-07-02T16:54:55 sp|P0DMQ6|DHSO_CHICK
528.8352051 0.974264706 0 (iTRAQ4plex)SANPATVPR 2 I K q-value XTandem_2016-07-02T05:20:51 sp|P12105|CO3A1_CHICK
697.3758545 0.899082569 0 (iTRAQ4plex)LPGPPGTSGNPGAK 2 G G q-value XTandem_2016-07-02T08:36:10 sp|P12105|CO3A1_CHICK
577.6469116 0.898450947 0 (iTRAQ4plex)GDVGLPGPPGPAGPAGDR 3 N G q-value XTandem_2016-07-02T08:36:10 sp|P12105|CO3A1_CHICK
483.6578674 0.861538462 0 (iTRAQ4plex)DGAPGT(Phospho)AGP 2 K I q-value XTandem_2016-07-02T11:41:38 sp|P12105|CO3A1_CHICK
858.0016479 0.904509284 0 (Glu->pyro-Glu)ELVVEQFEDLLVR 2 W I q-value XTandem_2016-07-02T06:23:44 sp|P13585|AT2A1_CHICK
534.8162842 0.853915663 0 (iTRAQ4plex)DKPPRSPK 2 M E q-value XTandem_2016-07-02T09:46:27 sp|P13585|AT2A1_CHICK
626.3561401 0.879604672 0 (iTRAQ4plex)DDEVSIHHR 2 E H q-value XTandem_2016-07-02T07:27:58 sp|P13648|LMNA_CHICK
626.3561401 0.879604672 0 (iTRAQ4plex)DDEVSIHHR 2 E H q-value XTandem_2016-07-02T07:27:58 sp|P13648|LMNA_CHICK
501.2928772 0.861538462 0 (iTRAQ4plex)DLLAAQAR 2 A I q-value XTandem_2016-07-02T11:41:38 sp|P13648|LMNA_CHICK
564.041687 0.984550562 0 (iTRAQ4plex)VLTVPLRRLLLPR 3 I I q-value XTandem_2016-07-02T10:40:59 sp|P15575|B3AT_CHICK
892.0001221 0.93289225 0 (iTRAQ4plex)KPITTIVSGGVEEVPL 2 K A q-value XTandem_2016-07-02T18:34:24 sp|P15989|CO6A3_CHICK
792.4298706 0 0 (iTRAQ4plex)EQEAAPAEEPAAAR 2 G Q q-value XTandem_2016-07-02T18:34:24 sp|P16527|MARCS_CHICK
792.4298706 0 0 (iTRAQ4plex)EQEAAPAEEPAAAR 2 G Q q-value XTandem_2016-07-02T18:34:24 sp|P16527|MARCS_CHICK
792.4298706 0.25 0 (iTRAQ4plex)EQEAAPAEEPAAAR 2 G Q q-value XTandem_2016-07-02T18:34:24 sp|P16527|MARCS_CHICK
792.4175415 0 0 (iTRAQ4plex)EQEAAPAEEPAAAR 2 G Q q-value XTandem_2016-07-02T06:23:44 sp|P16527|MARCS_CHICK
792.4175415 0.375 0 (iTRAQ4plex)EQEAAPAEEPAAAR 2 G Q q-value XTandem_2016-07-02T06:23:44 sp|P16527|MARCS_CHICK
792.4175415 0.904509284 0 (iTRAQ4plex)EQEAAPAEEPAAAR 2 G Q q-value XTandem_2016-07-02T06:23:44 sp|P16527|MARCS_CHICK
807.9940186 0.974264706 0 (iTRAQ4plex)HPRPLDGAAPNAAAL 2 G ] q-value XTandem_2016-07-02T05:20:51 sp|P17277|HXA4_CHICK
595.3776855 0.731034483 0 (iTRAQ4plex)QRHAKHLR 2 H V q-value XTandem_2016-07-02T11:41:38 sp|P18080|HEM0_CHICK
595.3776855 0.285714286 0 (iTRAQ4plex)QRHAKHLR 2 H V q-value XTandem_2016-07-02T11:41:38 sp|P18080|HEM0_CHICK
                             
                  (iTRAQ4plex)GM(Oxidation)VVGIKLDAGTAPLAGT 3 K N q-value XTandem_2016-07-02T08:36:10 sp|P07341|ALDOB_CHICK

 

2

Hi,

thank you for providing parts of your results. The q-value is defined as the "minimal False Discovery Rate at which a peptide with a given score would be accepted". It is a "local" property of a specific peptide not a property of the "global" full set of results, which usually makes it a better measure of confidence for a given peptide. That means, if a peptide (or PSM) has a q-value of 0.05, it first appears in the set of accepted peptides, when you set the FDR threshold to 0.05 (or higher). By default our FDR tool calculates this q-value and if you use IDFilter to filter the peptides, it will usually use the last score calculated for a peptide (unless specified otherwise). That is why your results have all q-values lower than 0.01 (with such a filter).

In case you are not familiar with the concept of a False Discovery Rate:

In our pipelines we usually use the target-decoy concept to simulate false discoveries. You include decoys in the database that you know are not present (e.g. reversed peptides from your original "target" database) and use the amount of identified decoy peptides as a proxy for the amount of falsely identified target proteins. The FDR then is only a measure of the proportion of falsely identified peptides in your overall accepted peptides (i.e. usually peptides above a threshold).

A small tutorial can be found here: http://www.inf.fu-berlin.de/lehre/WS14/ProteomicsWS14/LUS/lu7c/433/index.html

Cheers

Julianus

 

3

Hi Julianus,

Thank you for kind response and the tutorial part. What is the recommended level of threshold for peptide and proteins? Can you sugguest which one is good  either FDR score or q value for filtering false positives. In my case if i applying FDR score i am getting very very less number of PSM/proteins at 1% FDR score. 

4

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