Tutorial 3: Building a spectral library in Skyline

Transcription

1 SRM Curse 2013 Tutrial 3 Spectral Library Tutrial 3: Building a spectral library in Skyline Spectral libraries fr SRM methd design and fr data analysis can be either directly added t a Skyline dcument (in case a suitable library in the file frmats.sptxt,.blib,.hlf r.msp is available) r a custm library can be built within Skyline. Fr the latter, data-dependent acquisitin (shtgun) as well as SRM-triggered MS2 measurements searched with ne f the cmmn search engines can be used (currently 15 search engines are supprted). In this tutrial we describe hw yu can build a spectral library fr 30 synthetic peptides that have been measured in SRM-triggered MS2 mde n a QTrap instrument. 1. SRM methd preparatin fr SRM-triggered MS2 measurements Start with an empty Skyline dcument where the settings are already crrectly set: Open SRMcurse_ _Settings.sky frm the flder Tutrial-1_Settings and save it as SRMcurse_ _SRM-MS2.sky in flder Tutrial-3_Library. Adapt the settings as fllws: Peptide Settings: Keep settings as described in Tutrial 1 Settings, except fr the Library tab: Library: à N library shuld be activated. Transitin Settings Keep settings as described in Tutrial 1 - Settings, except fr the Filter and Library tabs: Filter: à Precursr charges: 2,3 à Prduct in charges: 1 à Prduct in types: y à Prduct ins frm: m/z > precursr, T: 2 ins à Always add N-terminal t Pr and C-terminal t Glu r Asp. à Precursr exclusin windw: 5 Th à Activate Aut-select all matching transitins Library: à Deactivate If a library spectrum is available, pick its mst intense ins Insert the list f peptides that were synthesised and fr which yu wuld like t select transitins t trigger an MS2 spectrum: Open target_peptides.xls in flder Tutrial-2_TransitinList, cpy the peptide sequences and paste them int Skyline: Edit à Insert à Peptides. Skyline autmatically inserts all peptides with the crrect prtein names and selects the 5 best transitins fr each precursr (light and heavy each). If all settings are setup crrectly, Skyline will autmatically insert the 30 target peptides under the crrect prtein name with selected transitins accrding t the filter definitin. In ttal yu shuld end up with a dcument cntaining: 408 transitins, 120 precursrs, 30 peptides and 10 prteins (see lwer right crner). As we want t trigger MS2 spectra nly frm heavy istpe-labelled peptides, all light transitins need t be remved: Edit à Refine à Advanced à Remve label type à select light 203 heavy transitins will remain. 1

2 SRM Curse 2013 Tutrial 3 Spectral Library Tip! D nt remve labelled peptides by deactivating the respective mdificatin in the Mdificatins tab in the Peptide Settings, because then Skyline des nt knw them anymre. (E.g. Skyline can nt use a library generated frm labelled peptides with a label type that is nt entered in the mdificatin tab.) Exprt this transitin list split int 2 methd files fr an AB Sciex instrument type File à Exprt à Transitin list: AB Sciex Multiple methds Max transitins per sample injectin = 103 Dwell time = 20 ms Activate the tab ignre prteins, which means that nt all peptides fr a given prtein will be measured in ne transitin list, but distributed amng different lists in case that wuld help t have less c-eluting transitins. In case, Skyline gives a ntificatin that the settings d nt match à N. Exprt transitin list as: SRMcurse_ _SRM-MS2.csv (the extensins 0001 and 0002 will be generated autmatically). Save the Skyline dcument. 2. Building a spectral library in Skyline After these tw SRM methds were run in SRM-triggered MS2 mde n a QTrap instrument, the files were searched with Sequest fllwed by PeptidePrphet as part f the trans-prtemic pipeline (TPP). Name f the tw SRM-triggered MS2 files: lgas_q130517_hdp_1.wiff lgas_q130517_hdp_2.wiff Name f the TPP utput file: interact_hdp_p0-9.pep.xml Imprt the.wiff files int t see the SRM traces that were used t trigger the MS2 spectra. File à Imprt à Results à Add ne new replicate à Name: lgas_q130517_hdp à select lgas_q130517_hdp_1.wiff and lgas_q130517_hdp_2.wiff. 2

3 SRM Curse 2013 Tutrial 3 Spectral Library Nte! These runs can be merged because they are cmplementary, i.e. n transitins were measured in bth injectins. Build a spectral library frm the acquired SRM-triggered MS2 spectra. Settings à Peptide Settings à Library à Build Give yur library a name: Mtb_hDP_ (hdp = heavy Dirty Peptides) Specify the utput path where yur library shuld be saved. We recmmend placing it int the prject-independent Skyline flder (see Tutrial 1). D nt activate the Keep redundant library ptin, as we wuld like t get fr each peptide just the single best spectrum, which is autmatically selected based n the best identificatin scre (based n Dt-Prduct all vs. all). Chse a false discvery rate f belw 1%, which in ur case is represented by a PeptidePrphet cut-ff scre f 0.9. A lab authrity name needs t be given fr each library, say SRMcurse. The field Library ID is ptinal and by default filled with the library name. Click Next and Add directry t specify the Tutrial-3_Library flder where the search engine utput (interact_hdp_p0-9.pep.xml) as well as the SRMtriggered MS2 files (cnverted t mzxml frmat) are stred. Yur Build Library tab shuld nw lk like this: 3

4 SRM Curse 2013 Tutrial 3 Spectral Library Finish the library build. Activate bth libraries. Place the Mtb_prteme_library first, such that it has pririty ver the newly created Mtb_hDP_ library (Edit list à Select up r dwn ). Place the MS/MS spectrum tab next t yur SRM traces by drag-and-drp. At the tp f the MS/MS spectrum tab yu can select frm the drp-dwn menu, which library spectrum yu wuld like t see first (in case bth libraries cntain an MS2 spectrum fr the respective precursr). Fr nw, select the Mtb_hDP_ library. G thrugh all 30 target peptides and check the btained SRM traces as well as the quality f the MS2 spectra. Nte! Sme f the spectra in the Mtb_prteme_library cntain prfile data, and, therefre, d nt wrk s well with Skyline's in ranking. There is a 50-peak cut-ff impsed fr perfrmance reasns and this causes that in sme spectra the peak picking is nt ptimal. 3. Selecting the mst intense transitins based n spectral library infrmatin Remve the SRM-triggered MS2 data frm the current dcument: Edit à manage results à select lgas_q130517_hpd and remve. Remve all peptides frm the left windw s that yu end up with a blank dcument. This is necessary t get rid f thse precursr charge states that are nt present in the libraries but that were selected with the filter settings used abve. Change the transitin settings as described in Tutrial 1 Settings: 5 mst intense y-in transitins, singly and dubly charged Dubly and triply charged precursr ins. Insert again the 30 peptides frm file target_peptides.xls (in flder Tutrial- 2_TransitinList): Edit à Insert à Peptides à Paste the peptide list. Skyline autmatically inserts all peptides with the crrect prtein names and selects the 5 best transitins fr each precursr (light and heavy each). Yu will end up with a transitin list f 449 transitins, 96 precursrs, 30 peptides and 10 prteins. Exprt a transitin list based n the new settings: AB Sciex, Multiple methds, Ignre prteins, Max transitins per sample injectin: 100, Dwell time: 20 ms, Name: SRMcurse_ _MtbPrtemeLib_hDP_Qtrap.csv. à Yu will get 5 transitin lists. 4

5 SRM Curse 2013 Tutrial 3 Spectral Library Save the Skyline file as SRMcurse_ _MtbPrtemeLib_hDP.sky. Tip! Yu can visualise and brwse all peptides f yur library in the spectral library viewer (View à Spectral Libraries): Exercises 1. Fr which peptides did the custm-made SRM-triggered MS2 library nt lead t an identified SRM spectrum? What culd be the reasns fr this? What culd yu d t get MS2-infrmatin? 2. Hw can yu als include b-ins int the autmatic transitin selectin? Hw many b- ins shw up amng the best 5 transitins? 3. What are the reasns fr the discrepancy in the number f transitins that are picked fr the light and heavy peptide versins? 4. Manually curate all peptides with less than 5 transitins. Hw d yu chse which ther transitins t add? 5. In file SRMcurse_ _SRM-MS2, why d sme precursrs have >2 transitins? 6. Fr SRM-triggered MS2, why d we select transitins abve the precursr and always add transitins N-terminal t Pr and C-terminal t Glu/Asp? We wuld like t thank Prime-XS and SystemsX fr supprting the SRM Curse