- Convert vendor specific mass spectrometry raw data into the mzML format using MSConvert31.
i. Open software tool msConvert.
ii. Select the file(s) to be converted.
iii. Select the output directory; if not specify, output file would by default be placed in the same directory as the input files.
iv. Choose "mgf" as “Output format”.
v. Only if the MS2 spectra are acquired in profile mode, select "peakPicking" as a filter. Otherwise, leave the “filter” option empty.
vi. Start the process by click on the "start" button.
CRITICAL STEP A detailed tutorial for such process can be found at (http://proteowizard.sourceforge.net/IntroductoryTutorial.shtml)
Generating Skyline input. ssl files
Linearize the sequences of identified cross-linked peptides as demonstrated in Fig. 2. This process can be automated using a combination of in-built functions in Microsoft Excel.
Generate .ssl files
A. Label-free quantitation
Convert modifications in the linearized sequences of cross-linked peptides into Skyline format (Fig. 1). A list of identified features of cross-linked peptides in the standard .ssl format
(https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BiblioSpec%20input%20and%20output%20file%20formats) can be generated using Microsoft Excel. Save this list as a text (tag delimited) file, and rename the ==*==.txt file as ==*==.ssl in file explorer.
B. SILAC based quantitation
Generate .ssl files as described in 3A. In the .ssl files, if a feature is identified as the non-isotope-labelled version of a cross-linked peptides, replace the sequence with the corresponding heavy (isotope-labelled) version.
C. Quantitation using isotope-labelled cross-linkers.
Generate a .ssl file containing all identified feature of cross-linked peptides as described in 3A. Generate a separate .ssl file containing pseudo identification information of cross-linked peptide features.
CRITICAL STEP This procedure requires co-elution of the light and the heavy versions of cross-linked peptides in the reverse phase chromatography in the LC-MS system. It is the case for BS3 and BS3-d4, however cross-linkers labelled with more deuterium atoms may lead to shift on elution time compared to their non-labelled analogues.
Quantitation using Skyline MS1 full scan filtering
Start Skyline and open a new file
Set parameters that are relevant to MS1 full scan filtering
i. Define modifications (Fig. 5)
==*== Select modifications that are predefined in Skyline
Select modifications from the dropdown list, such as Carboxymethyl (C) and Oxidation (M).
==*== Define user customized modifications
Settings>Peptide settings>Modifications>Edit list>Add
Table 1 lists modifications defined for BS3 cross-linking.
==*== Set "Max variable mods" according to input data.
==**== For SILAC based quantitation define isotope labelling scheme (*Fig. 6)
Settings>Peptide settings>Isotope modifications>Edit list>Add
Select predefined isotope modification from the dropdown list or defined customized isotope modifications.
ii. Peptide filters
"Min length", “Max length” and should be set accordingly to include all identified peptides that need to be quantified in the peptide library.
iii. Transition full scan parameters (Fig. 7a)
Settings>Transition settings>Full scan
==*== "Isotope peak included" = Count
==*== "Peaks" = number of isotope peaks used for quantitation.
CRITICAL STEP At least three isotope peaks are recommended for use in quantitation. However, this number should be less than the mass difference between the light and the heavy version of a cross-linked peptide when isotope-labelled cross-linkers are applied for quantitation.
==*== "Precursor mass analyzer" = analyzer applied for acquiring MS1 spectra.
==*== "Resolving power" = resolution applied for acquiring MS1 spectra.
CRITICAL STEP Precursor mass analyzer and Resolver power should be defined based on the mass spectrometer and acquisition method used.
==*== "Retention time filtering", select “use only scans within (X) minutes of MS/MS IDs”. The default time-window is set to five minutes, however it can be customized.
iv. Transition filter (Fig. 7b)
==*== "Precursor charge" should include all identified charge state of cross-linked peptides.
==*== "Ion type" = p (for precursor)
- Build a peptide library (Fig. 8)
Select the .ssl files as "input files".
CRITICAL STEP A separate library should be built for pseudo identifications of cross-linked peptide features when use isotope-labelled cross-links for quantitation.
- Select spectra libraries for quantitation.
==*== Select all spectra libraries that should be used for quantitation for a given dataset from the list.
==*== Select "Pick peptide matching library"
- Add peptides in the libraries into the list for quantitation (calculating transitions for quantitation).
==*== View spectra libraries in the spectra library explorer
==*== Select "Add all" to add all peptides in the library to the target list for quantitation. Based on the transition parameters set in Step 5, transitions are calculated for each cross-linked peptide. For SILAC labelled sample, transitions are calculated for both non-labelled and SILAC labelled version of every cross-linked peptide.
CRITICAL STEP If input data consists of multiple libraries (for example, a separate library is built for pseudo identifications of cross-linked peptide features when use isotope-labelled cross-linkers), after adding peptides from one library, switch view to another library using the dropdown menu and add peptides. Repeat the process until peptides in all libraries are added.
- Import mass spectrometry raw data for quantitation.
Upload data in .mzML format.
CRITICAL STEP For label-free quantitation, MS data of all conformers (including replicates) should be uploaded and quantified together. Similarly, when quantifying using isotope-labelled cross-linkers or SILAC, MS data from the forward-labelled and the reverse-labelled experiments should be uploaded and quantified together.
- Quality control of automated quantitation results.
CRITICAL STEP Like any other software, errors do exist in the automated quantitation results from Skyline which are mainly resulted from incorrectly isolated MS1 signals. This happens particularly more often to those low intensity signals. Skyline provides very user friendly interface for manually inspecting, and correcting the isolate peptide signals for quantitation. This process can dramatically improve the accuracy of quantitation results.
A. In a typical example, it takes less than 20 seconds to correct isolation windows of a cross-linked peptides quantified across six samples (two conformational states, each with triplicated MS analysis). Therefore, it is feasible to carry out manual inspection for every cross-linked peptide when quantifying dataset of few hundred cross-linked peptides.
i. View>Arrange Graphics>Tiled
Display chromatogram of all quantified samples in a tiled view (each MS acquisition in a separate window).
ii. Click on one cross-linked peptide listed in the "targets" list to display the extracted MS1 signals of this peptide in each MS acquisition.
iii. Right click on one chromatogram, under "peptide ID times" option select “matching” and “aligned” to display retention time of identified MS2 events and/or aligned identification of this peptide in each MS acquisition.
iv. View>Peak areas>Replicate comparison.
The abundance of isolated MS1 signals of the selected peptide in each MS acquisition are shown in a bar chart with isotope dot products labelled (in a new window). The isotope dot product compares the intensities of isolated isotope peak signals against the theoretical isotope abundance distribution calculated based on peptide sequence. A dot product value can range between 0 and 1, and 1 indicates a perfect match.
v. Inspect the isolated peptide signal according to following criteria:
==*== Isolation window includes the whole elution peak.
==*== Isolated elution window agrees with the retention time (RT) of identified MS2 events or aligned identification RT.
==*== Isotope dot product value is 0.9 or higher
==*== If Isotope dot product value is lower than 0.9, exam the MS1 spectrum of the selected peptide by clicking on the elution peak in the chromatogram (a new window will appear with MS1 spectrum).
vi. Correct the isolation window if not correct.
vii. Move to the next cross-linked peptides until going through all peptides in the "target" list.
B. When working with larger datasets, it becomes unfeasible time-wise to manually inspect quantitation results of each individual cross-linked peptide. In such cases, one could utilize Skyline’s inbuilt measurements for isolating peptide signals to filter out cross-linked peptide that are likely to be incorrectly quantified and carry out manual inspection and correction only on this subset of data.
i. Export the automated quantitation result following Step 11 and 12 with two additional data columns:
==*== Isotope Dot Product (Proteins>Peptides>Precursors>Precursor Results)
==*== Peptide Retention Time (Proteins>Peptides>Peptide results)
ii. View the automated quantitation results in Microsoft Excel (version 1997 and higher).
iii. Any isolated MS1 signal of a cross-linked peptide with isotope dot product value below 0.9 should be manually inspected.
iv. For label-free quantitation, calculate the coefficient of variation (CV) value on "Normalized Area" for the triplicate of each sample as standard deviation divided by mean. Cross-linked peptides with CV% higher than 20% within a sample should be manually inspected
v. For SILAC based quantitation, calculate the retention time difference between the light and the heavy signals of a cross-linked peptide. If it is larger than 20 seconds, manually inspect the isolated signal of this cross-linked peptides
vi. For quantitation using isotope-labelled cross-linkers when the retention time difference between the light and the heavy signals of a cross-linked peptide is larger than 40 seconds, manually inspect the isolated peptide signal
vii. Follow the procedures list in Step 10A, manually inspect the isolate MS1 signals of cross-linked peptides if it fulfils any conditions listed above (iii-vi)
viii. Export manually corrected quantitation results following Step 11 and 12.
- Customize contents (data columns) in the quantitation results report
View>Document Grid>Views>Edit view
Following data columns should be included in the results report.
==*== Peptide Modified Sequence (Proteins>Peptides)
==*== Replicate Name (Replicates)
==*== Normalized Area (Proteins>Peptides>Peptide results>Quantitation)
==*== Isotope Label Type (Proteins>Peptides>Peptide results) (only for SILAC based quantitation)
==*== RatioLightToHeavy (Proteins>Peptides>Peptide results) (only for SILAC based quantitation)
- Export report for automated quantitation results
The reports are in .csv format.