1. Workflows to set up acquisition methods for scheduled sMRM-HR on the TripleTOF 5600 Start from a data dependent acquisition (DDA) Perform data base search of your choice, Generate spectral library in Skyline Populate peptide tree in Skyline with your peptides of interest (from that library) that you wish to do sMRM-HR with For scheduled MRM-HR you will need to know the retention times for the peptides you will want to monitor so you can set up the scheduling. You can use the following different approaches for that, 1) scheduling from DDA files (through MS1 Filtering), 2) scheduling from SWATH files, 3) scheduling from multiple non-scheduled MRM-HR files. Several of the different possibilities are described below in the next slides (option 1 and 2), the principle and goal is the same. All you need to do is get a master file that was imported into Skyline that contains proper retention times for your peptides of interest - whether that comes from option 1 (DDA file), 2 (SWATH file), or 3 (non-scheduled MRM- HR runs); either will work setting up the scheduled sMRM-HR acquisition method.

2. 1) Take your DDA acquisition directly and import wiff file via MS1 Filtering (see MS1 tutorial ) into Skyline (your spectral library should be in Skyline prior to file import). Make sure you have 1 DDA file that will be your ‘master file’, for which all your peptides of interest have the correct peak picked and now the correct retention time assigned in the Skyline document. That is the file you later will use (point to) when making the acquisition method 1) scheduling sMRMHR methods from DDA files (through MS1 Filtering) The next slide shows a DDA file imported into Skyline You can see the precursor ion peak areas for M, M+1, M+2 The peptide tree is already populated with fragment ions (according to filters that add in the fragment ions from the spectral libraries for the individual peptides). However as this is a DDA file (and not a targeted, data independent MS2 acquisition) you do not see peak areas for the fragment ions yet, only the precursor ions. Once you have recorded your sMRM-HR runs later (after method acquisition) then you will see peak areas for these fragment ions after import of those files (SEE LAST SLIDE). fragment ions/ as well as precursor ions can be entered into the Skyline tree via File - Transition Settings, Filter

3. Data dependent (DDA/IDA) acquisitions, MS1 Filtering and Quantitation, 1x WT, 1x mutant K.SGSGTLTVSNTTLTQK.A Precursor ions M, M+1, M+2 Real and projected ID lines (DDA)

4. 1) scheduling sMRMHR methods from DDA files (through MS1 Filtering) Trim down DDA file and peptide tree to only the peptides you want to monitor for sMRMHR Confirm correct peak picking and RT in your master DDA file You can directly generate your sMRM-HR run now from this DDA file However the next slide would show you how to schedule if you had a SWATH acquisition (option 2), if you don’t have a SWATH file just ignore the SWATH description. However the scheduling sMRMHR steps described in the next few slides for scheduling from SWATH runs would be the same as if you scheduled from a DDA acquisition (so continue those steps when they are described).

5. Data independent SWATH acquisitions, 3x WT, 3x mutant, MS2 Quantitation K.SGSGTLTVSNTTLTQK.A –Fragment ions y3-y12 Projected ID lines (from DDA expmts) 2) scheduling sMRMHR methods from SWATH files

6. Workflow slide A Use SWATH runs to generate sMRM-HR mass spec method/peptide selection Take the SWATH Skyline file with all the replicate runs that had monitored about 1000 proteins from a protein lysate Reduce the Skyline peptide tree to only the peptides of interest to be monitored in the sMRM-HR experiment (here 419 peptides, 482 precursors) Take one SWATH acquisition replicate as ‘Master’ run to use for scheduling, make sure that for every peptide the correct acquisition time/ the correct peak is picked (Workflow slide B) If you have more than one file in your Skyline document upon method generation Skyline will ask for what file you want to schedule from (which one is your master file). Do a test to assess what the best time window width for scheduling is by estimating concurrent transitions for different time windows in the Skyline RT scheduling view (Workflow slide B, panel at the right) Export acquisition method from Skyline (File, export, method) - (Workflow slide C); point to an Analyst method template that contains all instrument parameters …(Workflow slide D). The newly generated method will then contain the scheduling list exported from Skyline (as defined from peak picking of the ‘Master’ file used for scheduling) (Workflow slide E) – also see final method setup in Analyst (Workflow slide F).

7. Workflow slide B Retention time scheduling view Retention time scheduling view can estimate how many concurrent precursor transitions there will be per what scheduling time window 1 transition per peptide estimated Scheduling with 2 min time window (blue line) appears to give reasonable concurrent transitions (<50) Important set up Right mouse click to obtain this view File – View – RetentionTime - scheduling

8. Method Export from Skyline File - Export - Method Definition of the scheduling window time Predefinition in Skyline for scheduling time width Workflow slide C This is your Analyst template method (that contains generic instrument settings, spray voltage, Gas1, temperature settings…)

9. MRM HR Analyst Template Method (used for Skyline as method template) This will be overwritten by what was defined by the user in Skyline (RT window width and m/z inclusion list). Scheduling width was defined in Skyline see workflow slide C on the left. This ‘dummy’ inclusion list must contain one m/z value here and the intensity 1 cps. Upon method generation this will be populated and the intensity setting will be automatically adjusted to ‘0’, and the RT window width will be also adjusted. Workflow slide D

10. New scheduled MRM HR method With populated list of peptides (m/z and RT) to be monitored for sMRM-HR Workflow slide E

11. The Analyst template method is basically a DDA or IDA method (see next slide) (even though in the end this will be a data independent acquisition method) You have 1 MS1 scan (250 msec) then product ion scans (max number of candidates times product ion accumulation time) However the IDA MS/MS triggering threshold is set so high 2000000 (next slide blue circle) that nothing will be triggered through that typical IDA mechanism. But MS/MS will be triggered through the inclusion list (that was added in by Skyline for the peptides in the peptide tree). As the inclusion list intensity threshold is set to 0 after Skyline generates that sMRM-HR method, the inclusion list will trigger the data independent MSMS acquisition, based on RT defined in the inclusion list and scheduling window width defined (here 120 sec). Setting up the template method initially: Max Candidates should initially be set to max concurrent peptides (see workflow slide B), upon Skyline method generation that value, (here 50) may be adjusted / fine-tuned to the proper value. Estimate your cycle time: if you have a max number of candidate ions per cycle say 50 then adjust your accumulation time for product ion scans so you get reasonable cycle times: here [1x MS1 scan]=250 msec plus [50x MS2 scans with accumulation time 60 msec] = 3000 msec Total cycle time = 3.3 sec Details see next slide

12. Scheduled MRM HR Workflow Acquisition Method Looped MS/MS Max candidates X for MS/MS accumulation time defines cycle time (+ TOF MS accumulation time) Max Candidates should initially be set to max concurrent peptides (see workflow slide B), upon Skyline method generation that value, here 50 may be adjusted / fine-tuned to the proper value. Adapted from Christie Hunter sMRM HR method in Analyst 50 0.060 sec 120 3.3 sec Values in red, parameters taken for my particular sMRM-HR experiment Workflow slide F

13. Setup of Skyline for file import of sMRM-HR files Either use full chromatogram import Make sure your peptide tree now is populated with fragment ions based on spectral library fragmentation and “File - Transition Settings – Filter Tab and Library Tab” settings Note: you can search sMRM HR data sets through regular search engines, then build a spectral library in Skyline, activate the library, then import sMRMHR runs, then the peak picking upon import will be directed by the MSMS in the spectral library

14. K.SGSGTLTVSNTTLTQK.A Data independent sMRM-HR acquisitions, 3x WT, 3x mutant, MS2 Quantitation Fragment ions y3-y12 Real and projected ID lines (DDA) sMRM-HR Results would look as follows Note you can search sMRM HR data sets through regular search engines