SRM Curse 2014 Tutrial 5 - Scheduling Tutrial 5: Retentin time scheduling The term scheduled SRM refers t measuring SRM transitins nt ver the whle chrmatgraphic gradient but nly fr a shrt time windw arund a peptide s expected retentin time. Hereby the number f measurable transitins per SRM run can be significantly increased. The mre precise retentin times f peptides can be predicted, the narrwer a retentin time windw can be defined and the mre peptides can be measured in a single run withut lss f sensitivity. Hence, peptide retentin time predictin is very imprtant fr targeted prtemics. Within Skyline, peptide retentin times can fr example be predicted using biinfrmatic algrithms, such as SSRCalc (Krkhin et al., Analytical Chemistry, 2006, 78, 6265), which estimate retentin times frm the amin acid cmpsitin and sequence f a peptide (mdificatins can s far nt be cnsidered). Hwever, the precisin f predictins with biinfrmatic tls is limited. Imprved values can be btained using empirically determined reference retentin times (irts, Escher et al., Prtemics, 2012, 12, 1111). In the fllwing tutrial we will shw yu hw t d retentin time predictin with SSRCalc and irts. 1. Theretical retentin time predictin with SSRCalc Open SRMcurse_20140210_MtbPrtemeLib_hDP.sky in flder Tutrial-3_Library and save it as SRMcurse_20140211_SSRCalc.sky in flder Tutrial-5_Scheduling. The file shuld cntain 450 transitins. G t Settings Peptide Settings Predictin Retentin time predictr Select frm the drp-dwn list Add. Set a retentin time predictr name: SSRCalc_SRMcurse Click the buttn Calculate >> in the lwer right crner. Open in Excel the file SSRcalc_calibratin_peptides.xlsx (in flder Tutrial- 5_Scheduling) and cpy-paste the peptide sequences and retentin times int the table Measured peptides. Nte! This Excel file cntains 12 f ur target peptides whse retentin times have been measured in a survey experiment under the same LC cnditins which will be used fr the main SRM measurements later n. Accurate retentin time infrmatin fr a small number f randm peptides (ideally spanning the whle retentin time range) is necessary t calibrate the SSRCalc-predicted retentin times t yur actual LC system. Cautin! The measurement frm which this retentin time infrmatin is taken shuld be carried ut immediately befre the main SRM measurements in rder t avid retentin time shifts due t clumn aging, clumn exchange, temperature changes, etc. Tip! It des nt matter which peptides yu use fr calibratin and yu can wrk fr example with a subset f yur endgenus target peptides (as in ur case) r synthetic reference peptides spiked int sample backgrund. Fr a gd calibratin curve, hwever, at least 10 peptides with accurate retentin times are required. Ideally, these peptides span the entire retentin time range. Tip! In case yu have imprted the data file f yur calibratin run int the Skyline dcument yu can als directly insert retentin time results via the Use results buttn (we will use this functin fr the irt calculatr, see belw). Select SSRCalc 3.0 (300A) frm the Calculatr drp-dwn list. 1
SRM Curse 2014 Tutrial 5 - Scheduling Skyline will nw generate a calibratin curve based n the 12 peptides yu pasted. The Slpe (0.825) and Intercept (-0.234) f the calibratin curve are filled in autmatically. The peptide number (12) and the R value (0.9653) are shwn right abve the table. Set the Time windw t 4 minutes Yur Edit Retentin Time Predictr tab shuld nw lk like this: Visualise the calibratin curve by clicking n Shw graph, clse it afterwards. Accept the retentin time predictr with OK. In Peptide Settings Predictin : Make sure that the abve defined retentin time predictr SSRCalc_SRMcurse is activated. Use measured retentin times when present shuld be activated (default) and the time windw shuld be set t 4 minutes. Accept the Peptide Settings with OK 2
SRM Curse 2014 Tutrial 5 - Scheduling Save the Skyline file (under the name which has already been defined abve). Nw that yu have activated a retentin time predictr, Skyline can assign expected retentin times t all target peptides defined in yur dcument. Skyline prvides a graphical view shwing the number f transitins which will be cncurrently measured depending n the selected retentin time windw size. T shw this graph g t: View Retentin times Scheduling Right-click n the graph and select Prperties t add mre time windws, fr example: 1, 2, 3, 4, 5, 10, 20 minutes. Explre the effect f the windw size nt the number f cncurrent transitins. Clse this Retentin Times windw befre cntinuing. Nw exprt a single scheduled transitin list. Fr this g t: Single methd, Optimizing = Nne, Methd type = Scheduled. Nw click OK. Skyline will shw the fllwing message: File Exprt Transitin list Select instrument type AB SCIEX, Select N t cntinue. Save the transitin list as SRMcurse_20140211_SSRCalc.csv in flder Tutrial- 5_Scheduling Inspect the transitin list in Excel. The third clumn nw cntains the predicted retentin time fr the current LC setup and n lnger the dwell time. Tip!: The scheduled windw size arund the predicted retentin time must be defined fr ABSciex instruments directly in the methd file n the instrument itself. Hwever, fr ther instrument vendrs, the scheduled windw size can be defined within the transitin list utput (cntains tw clumns, ne with start and ne with stp windw time, e.g. fr Therm). 3
SRM Curse 2014 Tutrial 5 - Scheduling 2. Empirical retentin time predictin using irt Start again with the Skyline file SRMcurse_20140210_MtbPrtemeLib_hDP.sky frm flder Tutrial-3_Library. Insert irt standard peptides int yur Skyline dcument (the nes we are using here are cmmercially available frm Bignsys). Open in Excel the file Transitin_list_iRT_peptides.xlsx (in flder Tutrial- 5_Scheduling) and cpy all fur clumns (peptide sequence, Q1, Q3, prtein) withut header. G t Edit Insert Transitin list paste the fur clumns. Nw yur Skyline dcument shuld cntain 472 transitins. Save the Skyline file in flder Tutrial-5_Scheduling as SRMcurse_20140211_iRT.sky. Imprt empirical retentin time infrmatin fr target peptides int Skyline. Imprt the data file arielb_q130619_017.wiff int Skyline: File Imprt Results Add single injectin replicates in file select arielb_q130619_017.wiff frm flder Tutrial-5_Scheduling. T generate this file, a pl f the 30 heavy reference peptides f ur case study was measured tgether with spiked-in irt peptides in nn-scheduled mde. Nte! Skyline will indicate a successful data uplad fr a given peptide/transitin by a clured circle in frnt f the respective peptide/transitin. The clur f this dt indicates the quality f the picked chrmatgraphic peak (green = gd quality, yellw = medium quality, red = bad quality), but is nt s reliable and shuld always be manually cnfirmed ne by ne. In a typical SRM experiment we wuld nw have t g thrugh the peptides ne by ne t make sure that always the crrect peak is picked. This prcess will be described in detail in Tutrial 6 - Manual Analysis. Because this sample was a pure mix f synthetic peptides, the peaks are all picked crrectly by Skyline and we d nt need t manually interfere. Tw windws (Peak Areas and Retentin Times) might pen, which yu can clse again. We will discuss these in Tutrial 6 - Manual Analysis. 2.1 Manually calculate irt values fr all target peptides The file arielb_q130619_017.wiff cntains the retentin time f all target peptides and the retentin time f the irt standard peptides. With this infrmatin we can cnvert empirical retentin times int unit-free irt values. Exprt the empirical retentin time f all targets and irt standard peptides by selecting: File Exprt Reprt select the default exprt called Peptide RT Results save as SRMcurse_20140211_iRT_reprt.csv in flder Tutrial- 5_Scheduling. Open the.csv file in Excel. Remve the clumns with the header ReplicateName, PredictedRetentinTime and PeptidePeakFundRati. Only PeptideSequence, PrteinName and PeptideRetentinTime are f imprtance fr us. Add tw new clumns called irt standard peptides and irt calculated. Int the clumn irt standard peptides paste the 11 irt values fr the standard peptides given in the Excel file irt_calibratin_peptides.xlsx, which can be fund in the flder Tutrial-5_Scheduling. Tip!: The Excel functin vlkup can be used t add infrmatin frm ne Excel table t anther ne. 4
SRM Curse 2014 Tutrial 5 - Scheduling Plt the empirical retentin times and irt values f the 11 standard peptides using the scatter plt functin. Label the axes with irt and Empirical RT. Right click n the data pints and add a linear trendline. Display the equatin and the R-squared value n the chart (under Optins ). Use the generated linear equatin t calculate irt values fr all yur target peptides in the clumn irt calculated. Yu did it the right way if fr the irt peptides, the calculated irt values are similar t the given irt values. Save the file as an Excel wrkbk (nt a csv file) with the name SRMcurse_20140211_iRT_reprt.xlsx. 2.2 Autmatically generate irt values within Skyline T create an irt calculatr in Skyline, g t Settings Peptide Settings Predictin. Click n the little calculatr icn and click Add t create a new irt Calculatr. Name: irt_srmcurse irt database: Click n Create and define the utput path f this calculatr. Name it irt_srmcurse and place it int the prjectindependent Skyline flder. It will be saved as an.irtdb file. Standard peptides: Cpy the peptide sequences and irts f the 11 irt peptides frm the file irt_calibratin_peptides.xlsx and paste them int the Standard peptides table. Tip! Here we use the irt peptides and crrespnding irt values which have been intrduced by Bignsys. Hwever, yu can als use any ther standard peptides and fix pints fr the calibratin using the buttn Calibrate right belw the Standard peptides table. Measured peptides: Click Add and select Add Results. Skyline nw cnverts all retentin time results frm yur current dcument autmatically int irt values (i.e. what we did manually in Excel befre). Cautin! It is crucial that the irt peptides were spiked int the sample, measured, and crrectly identified by Skyline. Cautin! If yu manually insert peptide sequences and irt values int the Measured peptides table frm an external surce by cpy-paste, make sure that all peptide mdificatins are indicated crrectly. Fr example carbamidmethylated cysteines need t be written as C[+57.0]. Tip! Via the Add buttn nt nly irt values frm results, but als frm spectral libraries can be added. This is, hwever, nly pssible if the spectral library cntains retentin time infrmatin fr target peptides AND standard (irt) peptides. The Edit irt Calculatr windw shuld nw lk like this: 5
SRM Curse 2014 Tutrial 5 - Scheduling Cnfirm yur irt calculatr with OK. Create an irt Retentin time predictr. In the Predictin tab f the Peptide Settings add a Retentin time predictr which uses the newly generated irt calculatr. T d s select frm the drp-dwn list f the Retentin time predictr Add. Name: irt_srmcurse Calculatr: select frm the drp-dwn list the irt_srmcurse calculatr we just generated. Activate Aut-calculate regressin. Cautin! Skyline will nw autmatically re-calculate, fr every data file that yu imprt, the calibratin curve based n the irt peptides. Be aware that this requires that thse standard peptides were present and measured in each run and that they were crrectly picked by Skyline, therwise significant errrs in retentin time predictin might ccur. Time windw: 4 min. Cnfirm the retentin time predictr by clicking OK. Activate the newly generated retentin time predictr irt_srmcurse by chsing it frm the drp-dwn menu in the Predictin tab f the Peptide Settings. Set the Time windw t 4 minutes. Accept the Peptide Settings with OK and save the Skyline file. Tip! T shw the predicted retentin time f each peptide directly within the chrmatgram windw, right-click n the data/chrmatgram windw and select Retentin time predictin (shuld be shwn by default). Nte that this is nly pssible if an irt value had been stred in the selected irt calculatr. The size f the yellw shaded area can be defined in Peptide Settings Predictin Retentin time predictr Drp-dwn menu: Edit current Time windw. Save the Skyline file. 6
SRM Curse 2014 Tutrial 5 - Scheduling 2.3 Exprt a scheduled transitin list using irt retentin time predictin The file arielb_q130619_017.wiff was used t extract retentin times f all ur target peptides and t stre them in the irt calculatr. Nw we d nt need this file anymre: Edit Manage Results Remve. Fr every scheduled SRM experiment we need an initial unscheduled calibratin run t determine the retentin times f the 11 irt peptides n the current instrument setup. This infrmatin can then be used t cnvert the stred irt values f all target peptides int expected retentin times n the current LC setup. Cautin! Fr such a calibratin run, the standard peptides have t be measured within the same peptide backgrund as the fllwing runs (yur samples f interest), because sample cmplexity can significantly influence peptide retentin time. Imprt the irt calibratin run int Skyline: File Imprt Results Add single injectin replicates in file select lgas_s130701_001_a1_rtcal.wiff Click thrugh the chrmatgrams f the 11 irt peptides. Inspect the linear regressin btained with the irt calculatr: View Retentin Times Linear Regressin. Right-click nt the linear regressin graph t switch between regressin curves btained with varius calculatrs. Save the Skyline file in flder Tutrial-5_Scheduling as SRMcurse_20140211_iRT_scheduled.sky Exprt a single scheduled transitin list fr the QTrap instrument by clicking: File Exprt Transitin list Instrument type AB SCIEX, Single methd, Optimizing Nne, Methd type scheduled. Save the transitin list as SRMcurse_20140211_iRT_scheduled.csv and inspect it in Excel. 7
SRM Curse 2014 Tutrial 5 - Scheduling Exercises 1. Cmpare the irt values that yu generated manually in Excel with the irt values generated autmatically within Skyline (yu can cpy-paste the Skyline values ut f the Retentin Time Calculatr table). 2. irt standard peptide GTFIIDPGGVIR seems t be an utlier in the retentin time linear regressin viewer f file arielb_q130619_017.wiff (in Skyline file SRMcurse_20140211_iRT.sky). Can yu explain what is the issue here? 3. What is the difference between a Retentin time calculatr and a Retentin time predictr? 4. Which retentin time windw size wuld yu apply if yu wanted t run a single scheduled methd with maximally 150 cncurrent transitins? Culd yu apply this t SSRCalc as well as irt predictin? 5. Cmpare the predicted retentin times frm SSRcalc and irt calculatr, generate a graph shwing the crrelatin between bth predictrs. Which crrelatin cefficient (R 2 ) is achieved? 6. In a scheduled SRM methd, nt the dwell time but the cycle time is kept cnstant. This means that the dwell time fr a given transitin can change ver time depending n the number f cncurrent transitins. T which minimal dwell time des this apprximately lead when using the transitin list generated abve with a 4 min retentin time windw size and a fixed cycle time f 2 secnds? At which time f the gradient des this minimal dwell time apprximately ccur? 7. Why d cntinuusly changing dwell times in scheduled SRM runs nt lead t bservable differences in signal intensities? (lnger dwell times shuld increase signal intensity, shrter dwell times decrease signal intensity) 8. What is the difference between SSRCalc 3.0 (100A) and SSRCalc 3.0 (300A)? We wuld like t thank Prime-XS and SystemsX fr supprting the Zurich SRM Curse 2014. 8