SRM Curse 2013 Tutrial 4 Parameters Tutrial 4: Parameter ptimizatin The aim f this tutrial is t prvide yu with a feeling f hw a few f the parameters that can be set n a QQQ instrument affect SRM results. Therefre, we will ask yu several times alng the tutrial t brwse thrugh all the peptides and bserve the effects f a certain parameter. Infrmatin abut the sample used: in this tutrial we used the same mixture f 30 crude and heavy synthetic peptides that was used as a reference in the previus tutrials. Such a synthetic peptide mixture allws ptimizatin f all instrument parameters frm a lw-cmplexity sample leading t ptimal results in terms f sensitivity and data quality. Further, the sample amunt f synthetic peptide mixture is typically very high, which fr parameter ptimizatin is imprtant, as this requires multiple injectins. A. Cllisin Energy Optimizatin (CEO) Nte: n the Skyline website yu can find a very detailed tutrial n CEO. The fllwing tutrial has been adapted t ur case study. CEO can be perfrmed fr several purpses such as t ptimize the general CE equatin fr yur instrument, t further ptimize CEs fr selected precursrs r t even ptimize CEs n transitin level. In the fllwing we perfrmed a CEO experiment fr the 30 heavy synthetic peptides used already previusly thrughut ur case study. Transitin Settings fr CEO In rder t prepare transitin lists fr CEO, yu must chse the ptimizatin parmaters in the Cllisin Energy drp-dwn menu: by hw much t increase the CE (step size), and hw many steps wuld yu like t include (step cunt). Nte that the final number f steps is defined as (step cunt*2 +1), as CE steps will be added abve and belw the deafult chsen value, which is als included. Open the file SRMcurse_20130715_Settings.sky, that was created yesterday. Peptide Settings: à Apply settings as described in Tutrial 1 - Settings Transitin Settings: à Apply transitin settings as described in Tutrial 1 - Settings, except fr the Predictin tab. Here select frm the Cllisin Energy drp-dwn menu: Edit list à select ABI QTrap 4000 à click Edit. An Edit Cllisin Energy Equatin windw will pen. In the lwer part f this windw yu can define the CEO step size (à select 2), and step cunt (à select 5). 1
SRM Curse 2013 Tutrial 4 Parameters Insert a transitin list int yur Skyline dcument We use a transitin list similar t the list yu generated in Tutrial 2 - TransitinList. The cmplete list is prvided in file transitin_list_fr_ceo.xlsx in flder Tutrial-5_Parameters. (reminder fr insertins: Edit à Insert à Transitin list à paste frm Excel) Yu shuld end up with a Skyline dcument cntaining 10 prteins, 30 peptides, 30 precursrs and 143 transitins. Save yur Skyline dcument as SRMcurse_20130716_parameter_settings.sky in the flder Tutrial-4_Parameters. Transitin list exprt fr CEO T perfrm CEO measurements, Skyline can autmatically exprt a transitin list including each transitin several times assciated with a different CE. T allw the same transitin t be cncurrently measured and analyzed, the Q3 value fr each step needs t be slightly changed. T exprt a transitin list autmatically including all CEO parameters g t: File à Exprt à transitin list à select: AB Sciex, single methd, Optimizing = Cllisin Energy. Click OK, and click N when asked if t use default (meaning that in this case we d nt want t use the default). Save this transitin list as SRMcurse_20130716_CEO.csv. Open the file in Excel. Yu will nte that there are mre than 1500 transitins generated (143*11). As these are t many transitins t be measured in ne methd, we had split this transitin list and measured it in several injectins. Yu d nt need t d it nw. Nte: In future, if yu perfrm CEO, yu shuld aim t measure the CEO transitins f a precursr in the same run. Therefre, in case yu split yur transitin list int several methds, accunt fr the step number in yur calculatin f transitin number per injectin, which shuld be #transitins * #steps, t ensure that CEO transitins fr a precursr are measured cncurrently. 2
SRM Curse 2013 Tutrial 4 Parameters Imprt raw files int yur CEO-Skyline dcument Imprt fur measurement files int the Skyline dcument: File à Imprt à Results à Add ne new replicate à name it CEO à frm the Optimizing drp-dwn menu chse Cllisin Energy à select the 4 measurement files called: CEO_1.wiff, CEO_2.wiff, CEO_3.wiff, CEO_4.wiff Nte: These files were measured in scheduled SRM mde, a tpic which will be cvered later in the curse. Frce Skyline t integrate all transitins by selecting: Settings à Integrate all Adjust yur view settings: View à Transitins à Single; View à Peak Areas à Replicate cmparisn; View à Aut-zm à Best peak; (Optinal: View à MS/MS spectrum (t view the library)). Yur Skyline dcument shuld nw lk like this: 3
SRM Curse 2013 Tutrial 4 Parameters In the CEO windw, each trace represents the transitins measured in a single CEstep, and the matching peak area is presented in the Peak Areas windw. These are centerd arund the default value (in red). Brwse thrugh all peptides and check ut the effect f CEs nt signal intensity. Brwse thrugh the transitins f several peptides and inspect the the effect CEO has n the transitin level (gd example: TTIYDLHGASQGITR). Nte: CEO will affect the intesity f each transitin in a different manner! Save this Skyline dcument as SRMcurse_20130716_CEO.sky. Effect f CEO nt the relative transitin intensities per peptide Since CEO affects transitin intesities differently it can als implicate relative intesities, which cnsequently must be taken int accunt when cmparing SRM traces t a library MS2 spectrum. T demnstrate this carry ut the fllwing steps: Imprt tw new results as single replicates (CE_plus10.wiff and CE_minus10.wiff). As the names suggests, these files were acquired with a plus/minus difference f ±10 V. Arrange the windws s that yu can see them all side by side. Change yur view t see all transitins (View à Transitins à All). Change the y-axis in the peak area windw by nrmalizing all transitin intensities t the ttal intensity: right click n the windw à nrmalize t à ttal intensity Nw brwse thrugh all peptides and examine the impact f CE n relative intesities: while fr sme peptides we saw that CEO imprved signal intensities, it can als result in mdified relative intesities. T further exemplify this, we can lk at the change in the dt prduct (dtp). T visualize it : right click n the windw Peak Areas à nrmalize t à Nne; inspect the change in dtp. In the next few days we will use relative intensities and dtp in chsing the crrect peak. Therefre, remember that these are als affected by CE. Save this Skyline dcument as SRMcurse_20130716_CE_10minus10.sky. Remve the tw result files CE_plus10.wiff, CE_minus10.wiff: 4
SRM Curse 2013 Tutrial 4 Parameters Edit à Manage results à Select these files à Click Remve. Make sure t leave the file CEO. In case it disappeared frm the view: View à Arrange graphsà Tiled. Exprt a new transitin list with ptimized CEs Yu can exprt transitin lists with ptimized CEs either n peptide r transitin level by activating the crrespnding check bx: Settings à Transitin Settings à Predictin à activate use ptimizatin values when present à ptimize by = transitin. Exprt nw a transitin list and name it SRMcurse20130716_ptimizedCE.csv. G back, deactivate this check bx, exprt anther transitin list SRMcurse20130716_defaultCE.csv. Generate a custm-made CE equatin fr yur persnal QQQ instrument Measured CEO values can als be used t extraplate a new ptimal CE equatin fr yur QQQ instrument f interest. Fr this select: à Transitin settings à Predictin tab à Cllisin energy à select frm the drp-list Add à Use Results à t visualize the linear regressin select Shw Graph à Name the new equatin as QTrap_SRMcurse and activate it as the cllisin energy equatin f chice fr the dcument Hw des the new equatin cmpare t the ne prvided by the manufacturer? Save this Skyline dcument as SRMcurse_20130716_custm_CE_equatin. 5
SRM Curse 2013 Tutrial 4 Parameters Cautin! Newly generated CE equatins can be very sensitive t the peptides chsen fr ptimizatin (in this tutrial yu may get a different slpe. Als, if yu wish: unpick several precursrs and see what happens t the equatin). Hence, t generate a rbust and truly representative equatin it is advisable t wrk with a sufficiently large number f peptides, cvering the whle range f physicchemical prperties (ideally 100s f peptides). B) Declustering Ptential Optimizatin (DPO) The same wrkflw as described abve fr CEO can als be used fr Declustering Ptential Optimizatin (DPO). Step size and step cunt can be selected as fllws: à Transitin Settings à Predictin à Declustering Ptential à Edit list à select ABI à Edit à define step size = 2 and step cunts = 5 T visualize DPO measurements imprt the three files DPO_1.wiff, DPO_2.wiff, DPO_3.wiff (chse Add ne replicate à name it DPO à select in the Optimizing drp-list declustering ptential ). Arrange yur view t lk like this (again activate View à Transitins à Single 6
SRM Curse 2013 Tutrial 4 Parameters Nte: Here yu see side by side, the steps f CEO and DPO, which are labeled and clred similiary, althugh these are steps f different ptimizatin prtcls! Brwse thrugh all peptides and investigate the influence f CEO and DPO next t each ther Save this Skyline dcument as SRMcurse_20130716_CEO_DPO.sky. C) Dwell time/ Cycle time T explre the effects f increasing the cycle time we have measured the same transitin list at different dwell times, in an unscheduled mde (reminder: dwell time * #transitins = cycle time). Althugh we have increased the dwell time, the main take hme message f this part relates t increasing cycle time. 1. Imprt Dwell time results int the Skyline dcument Remve all the results related t CEO and DPO via Edit à Manage results à remve all Imprt the 5 files frm flder Tutrial-4_Parameters that are named unscheduled_dwell*ms.wiff with * standing fr 10, 20, 40, 60 and 100 ms. When asked, d nt remve the cmmn prefix. Brwse thrugh the peptides and examine the effect f dwell time nt the peak shape (fr example the peptide HLPEHAIVQFVK, r the peptide VIGVPAMFAAGDVAAAR). Nte the change in the shape f peaks, resulting frm increased cycle time (which means less pints per peak). Yur Skyline dcument shuld lk like this: 7
SRM Curse 2013 Tutrial 4 Parameters Skyline ffers several ptins fr smthing peaks (aiding in visualizatin and peak picking). In general, smthing relies n sufficient pints f measurements. Have a lk at the intensity f a peptide and then perfrm a visual smthing f the data by selecting: View à Transfrm à Savizky-Glay smthing. Nte that as the cycle time gets lnger (in this case because the dwell time gets lnger) we have fewer measurement pints and the intensity f the peak is changed cnsiderably after smthing. Imprtant: the peak areas d nt change after smthing, since smthing is nly affecting visualizatin. Save this Skyline dcument as SRMcurse_20130716_cycletime.sky 8
SRM Curse 2013 Tutrial 4 Parameters Exercises Lk at the fllwing transitin table fr CEO: 813.439 740.3425 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 31.1 813.439 740.3525 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 33.1 813.439 740.3625 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 35.1 813.439 740.3725 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 37.1 813.439 740.3825 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 39.1 813.439 740.3925 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 41.1 813.439 740.4025 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 43.1 813.439 740.4125 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 45.1 813.439 740.4225 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 47.1 813.439 740.4325 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 49.1 813.439 740.4425 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y8.heavy 51.1 813.439 669.3054 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 31.1 813.439 669.3154 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 33.1 813.439 669.3254 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 35.1 813.439 669.3354 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 37.1 813.439 669.3454 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 39.1 813.439 669.3554 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 41.1 813.439 669.3654 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 43.1 813.439 669.3754 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 45.1 813.439 669.3854 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 47.1 813.439 669.3954 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 49.1 813.439 669.4054 Rv1812c_Rv1812c.VIGVPAMFAAGDVAAAR.+2y7.heavy 51.1 Hw many steps were used? What is the step cunt? What was the step size used? What is the increment used fr Q3? Fr which peptide has CEO generated the mst significant imprvement in signal intensity? T clarify the use f Q3 increments, set the Match tlerance m/z t 0.01 (Settings à Transitin settings à Instrument). Hw many steps d yu see nw? Re-adjust the value t 0.055. 9
SRM Curse 2013 Tutrial 4 Parameters Since yu knw the dwell times we used fr each file, hw lng was the cycle time in each file? Given the cycle time, estimate hw many pints are measured n average. Althugh we have ptimized the CE per transitin, we will nt use it in the next tutrials. Why? Hmewrk: Further explre CEO by pltting the default CE and the ptimized CE per transitin in Excel. Use the tw transitin lists that yu exprted. Plt the default CE against the ptimized CE per transitin. Apprximately hw big is the difference? Hw culd this infrmatin affect the chice f step cunt and step size fr future CEO experiments? Hmewrk: Further explre relatinships between quantificatin and cycle time by lading the Results grid (in View) and brwsing thrugh the precursrs. Have a lk at the backgrund and peak areas fr example. Fr the interested curse participants the fllwing tpic can be cvered as well: D) SRM-triggered MS2 Fr SRM-triggered MS2 a list f transitins needs t be prvided t the QTrap instrument. This list can be prduced withut a priri knwledge (fr example selecting 2 y-ins per precursr) r with a priri knwledge (fr example, frm previus spectra). Fr detailed instructins see Tutrial 3 - Library. Fr this sectin, yu are prvided with tw lists f transitins. Start with the Skyline file SRMcurse_20130715_settings frm flder Tutrial- 1_Settings. Change the Min m/z t 250 (Transitin settings à Instrument). This transitin list was measured n a QTrap instrument in SRM-triggered MS2 mde. The crrespnding files were cnverted t mzxml and searched using SEQUEST. In the Tutrial-4_Parameters flder yu will find an SRM triggered MS2 flder which cntains mzxml files as well as a pep.xml file. Build a library frm these: Peptide settings à Library à click Build. Name this library Srmcurse_20130716_trigms2. KEEP the library redundant and use a cutff scre f 0.1. Click Next and then Add files and add the trigms2.interact.pep.xml file in the tutrial flder. Click Finish. 10
SRM Curse 2013 Tutrial 4 Parameters When building the library is cmplete, chse Library frm the Pick peptides matching drp-dwn menu. Insert the transitin list named transitin_list_fr_theretical_trigms2.xlsx. Save the Skyline file as SRMcurse_20130716_trigMS2.sky. Imprt as ne replicate (name it theretical_trigms2 ) the tw files trigms2_part1-arielb_mtb_dp_srm_curse_007.wiff and trigms2_part2-arielb_mtb_dp_srm_curse_008.wiff. Cautin! make sure yu lad the wiff and nt the mzxml files. In Transitin settings à Full scan à Leave the MS1 filtering as is. In the MS/MS filtering chse Targeted in the Acquisitin Methd and QIT as the Prduct mass analyzer. Click OK. Yu will nw be able t see when and where an MS2 event was triggered (marked as the lines labeled with ID). Yu can click n these t see the respective MS2 scans, r scrll thrugh these in the MS/MS spectrum windw: By brwsing thrugh the peptides yu will ntice large differences between the scans in intensity, peak shapes and number f IDs. Fr sme precursrs n MS2 spectrum is available, while fr thers there are quite a few spectra. When generating an SRM assay frm such an experiement we may want t be careful which spectrum is used. See fr an example the peptide AQAATAGIDDLRPALIR. Nte: Parameters, such as hw ften and at what thershld t trigger an MS2 can be tuned n an instrument. Once we have btained an MS2 spectrum fr each peptide, we can extract the list f tp six transitins and re-trigger the MS2 n these newly selected transitins, instead f theretical transitins. T exemplify this, add t the current transitin list the same transitins yu have used fr the CEO (transitin_list_fr_ceo.xlsx). The tw lists will be merged and yu will have a ttal f 284 transitins. Nw imprt the file trigms2_yins-arielb_mtb_dp_srm_curse_037.wiff as ne replicate. Arrange the windws side-by-side and brwse thrugh all peptides. 11
SRM Curse 2013 Tutrial 4 Parameters General ntes: Since the tw files d nt verlap cmpletely in transitin lists, sme precursrs/ fragment ins may be fund in ne but nt the ther. The newly imprted file shuld cntain a gd SRM trace fr each peak, during which several IDs are acquired: > This prcedure may enable us t avid errrs in chsing the crrect peak. See fr example the peptide AIVHTAAELVDAR r the peptide VTTSTGASYSYDR. > In certain cases, yu can ntice significant difference in intesities. See fr example the peptide HLIDDALK. > Since we have searched these files and generated a library frm these searches, the current visualizatin wuld be sensitive t changes in the parameters f this prcess (search paprameters, library building parameters etc). Furthermre, since these are un-purified (crude) synthetic peptides we expect t see IDs als at weird times alng the chrmatgram (ne reasn is that these peptides can still have prtecting grups which are lst nly during the sample analysis n the instrument). We wuld like t thank Prime-XS and SystemsX fr supprting the SRM Curse 2013. 12