SRM assay generation and data analysis in Skyline

Transcription

1 in Skyline Preparation 1. Download the example data from (3 raw files, 1 csv file, 1 sptxt file). 2. The number formats of your computer have to be set to English (United States). Open the Control Panel Clock, Language, and Region Change Location Formats English (United States). Skyline document set-up 1. Open Skyline. 2. Go to Settings Peptide Settings and go through all tabs: a. Digestion i. Enzyme: We use trypsin, which cleaves C-terminally of lysine (K) and arginine (R), but not if these are followed by a proline (P): select Trypsin [KR P] ii. Max missed cleavages: We work with fully tryptic peptides only: select 0. iii. Background proteome: None. b. Prediction i. We do not use these settings for our case study, keep the defaults. c. Filter i. Min/Max length: We work with peptides of 7 to 25 amino acids length. ii. Exclude N-terminal AAs: Enter 0. iii. Exclude potentially ragged ends: Do not select this option. iv. Exclude peptides containing: Do not select these options. v. Auto-select all matching peptides: Do not select this option. d. Library i. Libraries containing MS2 spectral information can be downloaded from public sources or built within Skyline. We are going to import an Mtb spectral library, which has already been published. Go to and click on the 4000 QTrap link to download and unzip the spectral library in.sptxt format. ii. Edit list Add iii. Name it e.g. Mtb_Qtrap, then select the downloaded.sptxt file and confirm. iv. Select the newly generated library. v. Pick peptide matching: Library. vi. Rank peptides by: We do not need this. e. Modifications i. Structural modifications: Structural modifications concern chemical modifications of peptides. They can either be static (always present) or variable (sometimes present, sometimes not). Add here Carbamidomethyl (C) as a static modification. It comes from the cysteine reduction and alkylation step during sample preparation. ii. Max variable mods and Max neutral losses: Keep the defaults. iii. Isotope label type: Leave the default heavy. iv. Isotope modifications: We spiked-in synthetic reference peptides of which the C-terminal Arg or Lys contain 13 C and 15 N atoms. Activate these modifications: Edit list Add: 1. Label:13C(6)15N(2) (C-term K) 2. Label:13C(6)15N(4) (C-term R) v. Internal standard type: Leave the default heavy. f. Click OK to confirm all peptide settings. 1

2 3. Go to Settings Transition Settings and go through all the tabs: a. Prediction i. Precursor mass and product ion mass: We work with the monoisotopic mass. ii. Collision energy: Keep the default. iii. Declustering potential: We do not apply a customised DP. iv. Use optimisation values when present: Do not select this option. b. Filter i. Precursor charges: We work with doubly and triply charged precursors ( 2, 3 ). ii. Product ion charges: We consider singly and doubly charged product ions ( 1, 2 ). iii. Ion types: We focus on y-ions ( y ). We do not consider b-ions, because compared to y-ions they tend to be less conserved between instruments. iv. Product ions: 1. From: ion 1 and To: last ion. 2. Always add: Do not select anything here, because we are going to use the library to guide the selection of product ions. 3. Precursor m/z exclusion window: 10 Th. v. Auto-select all matching transitions: Activate this option. c. Library i. Ion match tolerance: This depends on the instrument type that was used to acquire your library. Lower values help to get a more specific peak assignment of the spectra, but if the instrument doesn t have this accuracy you will loose your peaks. Enter a mass tolerance of 1.0 Th. ii. If a library spectrum is available, pick the most intense ions: Select this option and pick the 5 most intense transitions per precursor. iii. From filtered ion charges and types: Select this option. d. Instrument i. Min/Max m/z: We typically use 300 to 1350 m/z. ii. Dynamic min product m/z: We do not use this option. iii. Match tolerance: We keep the default setting of Th iv. Firmware transition limit: Do not enter a limit. v. Firmware inclusion limit: Do not enter a limit. vi. Min/Max time: Leave these fields empty. e. Full-Scan: We do not use this. f. Click OK to confirm all transition settings. 4. Save the Skyline document: File Save as MyFile.sky SRM assay generation in Skyline With SRM assay generation we mean the selection of the most intense transitions per peptide. The selection of the optimal peptides per protein had been done before. 1. To insert the peptide list into Skyline, copy the peptide sequences together with the protein names from the file TargetPeptides.csv. Back in Skyline go to Edit Insert Peptides and paste the copied peptides and proteins here. 2. Skyline now automatically groups the peptides into proteins and selects the highest 5 transitions for each peptide from the spectral library which we specified it in the transition settings before. 3. Go through the transition list and look at the corresponding spectra. a. In the lower right corner you can see the number of proteins, peptides, precursors and transitions you have in your Skyline file. 4. This is your assay Skyline file, save it as MyFile_assays.sky. 2

3 5. Export the transition list to feed into the instrument: File Export Transition List. a. Export an ABSciex transition list using the standard settings and a dwell time of 20 ms. b. A pop-up window asks you if you would like to use the defaults, say No. c. Save this transition list as MyFile_assays.csv. d. For the measurement we need precursor m/z, product m/z, dwell time, transition ID, declustering potential, and collision energy. How many proteins, peptides, precursors and transitions do you have in your Skyline file? For how many peptides do we have the 3+ precursor and for how many do we have the 2+ precursor in your Skyline file? Which is the predominant charge state of the fragment ions in your Skyline file? How many precursors are you left with, if you only select the 2+ precursors (change in Transition Settings)? Please change this setting back to 2+ and 3+ precursor before you continue. 3

4 SRM data analysis in Skyline After the transition list has been measured on a triple quadrupole instrument, we import the data into Skyline to check the quality of the runs and to quantify our target peptides. 1. Import SRM data: File Import Results Add single injection replicates per file OK select all raw files in the data folder Open a. Skyline will ask if you want to remove the common prefix of all files, select Do not remove to keep the complete file name. 2. To get a good overview over the data go to View Retention Times Replicate Comparison. And View Peak Areas Replicate Comparison. Then grap one of these new windows, drag them down to the little arrow pointing down, then release. Arrange all the other tabs likewise such that your Skyline looks like this: 3. This is your raw Skyline file, save it as MyFile_raw.sky. 4. Explore the SRM peak groups: a. Right click on the peak groups and play with the zoom to better see your peaks. b. You can see that the transitions have only been measured during 4 min (scheduled SRM). For time reasons we did not discuss scheduled SRM measurements in detail in this tutorial and therefore we will ignore this. But be aware that if we had measured a transition list without scheduling, we would have had for each transition a signal over the whole 30 min gradient. 5. Manually refine the peak picking: a. Make sure that the right peak is picked. If certain peaks are wrongly picked by Skyline, drag the area boundaries to what you think is the correct peak. b. The peak boundaries have to be the same for heavy and light (drag areas when you have the peptide activated, not just one precursor or a single transition). c. It is a good idea to look at the Retention Time and Peak Area windows from time to time, they help to spot problematic peaks. 6. Manually refine the transitions which should be used for quantification, i.e. remove low quality transitions by deleting them from the transition tree window. a. For correct quantification, the heavy and the light precursors need to have the same transitions. 7. This is your refined Skyline file, save it as MyFile_refined.sky. 8. To export the data to a.csv file: File Export Report 9. We will generate our own report format: Edit list Add 10. Give the newly created report a name and browse the tree view to find and select the following features: ProteinName, PeptideSequence, RatioToStandard. 4

5 11. Check the Pivot Replicate Name option, then save this report template. 12. Export and save your data in this report format: MyFile_refined.csv. Which transition of the peptide LLGSVSSGLLR is behaving weird? Does the removal of the two shouldered transitions of the peptide LPDGNGIELCR have an impact on the ratio between heavy and light? (see total ratio next to the precursor mass) Which transition of the peptide VIGPAMFAAGDVAAAR is not co-eluting with the rest and how does the dot product change if you delete it? Finalising SRM data analysis in Excel 1. Open your exported Skyline report in Excel. 2. Determine the relative changes of each peptide to the first time point. Define for the first time point the relative abundance = 1 (i.e. divide all ratios by the first ratio). 3. Average the peptide changes for each protein and plot the relative changes of the proteins over the 3 time points. 4. The highly regulated proteins prevent us to visualise the less regulated proteins. If you log-transform the y-axis (e.g. using log 2) you will see the following: a. Large changes are compressed and we can better see the less regulated proteins. b. Shifted the not regulated from 1 to 0 and rescales the down-regulated proteins such that they have the same scale as the upregulated just in negative numbers. Before, the ratios for down-regulated proteins were all squeezed between 0 and 1, while the ratios for up-regulated proteins went from 1 to. 5. The final graph should look similar to this: Ratio changes of 6 Mtb proteins during hypoxic stress Log2 (ratio to time point 0) h 6h 48h Rv2623 TB31.7 Rv3133c devr Rv0079 Rv0079 Rv2626c hrp1 Rv2027c dost Rv1812c Rv1812c Do the peptides belonging to the same protein show similar ratio changes? Discuss what could be the reason for peptides showing different regulation. 5