1. Software installation:
Download TDFragMapper by clicking on the Download button at https://msbio.pasteur.fr/tdfragmapper.
The following workflow demonstrates how to visualize and compare fragments obtained from various tandem mass spectrometry (MS/MS) experiments on intact proteins using TDFragMapper.
2.1. Execute the TDFragMapper tool (Figure 1)
Figure 1: Graphical User Interface of the main window of TDFragMapper.
2.2. Setting parameters
2.2.1. Protein Sequence: specify a file containing a single protein sequence. The file format can be txt or FASTA, obtained from Uniprot.
2.2.2. Sequence Information: specify any additional information that will appear with the legend of the fragmentation maps.
This field is not mandatory.
2.2.3. For each data file, specify a Fragmentation Method, an Activation Level, a Precursor Charge State, a Replicate and the path to the MS/MS Data file, which was generated by ProSight Lite (Figure 2). Specify a Deconvoluted Spectra file that was generated by Thermo® FreeStyle™ software (Xtract algorithm) if an evaluation of fragment ions intensity is desired.
18.104.22.168. To add new data file parameters, click on the Add another data file button. A new line will be included in the table.
22.214.171.124.1. Instead of filling file by file, the user has the possibility to fill multiple data files at once by copying from a table (e.g., Excel® file), and pasting by right-clicking on the table or pressing CTRL + V. The table (e.g., Excel® file) must respect the following format:
Table 1: Data to be copied and pasted on TDFragMapper.
The following nomenclature (commonly used in tandem mass spectrometry experiments) has to be followed in order to standardize the writing of Precursor charge state, Activation level, Replicate, and Fragmentation method:
Fragmentation method: CID; SID; HCD; UVPD; EThcD; ETD; ECD;
CID, SID, HCD: … % (e.g.: 25%)
UVPD, ECD & ETD: … ms (e.g.: 70 ms)
EThcD: … ms /… % (e.g.: 10 ms / 10 %)
Precursor charge state: only numbers (e.g.: 25)
Replicate: R… (e.g.: R1, R2, R3, etc.)
126.96.36.199.ProSight Lite: For generating an output file, after opening a *.pcml file (or setting up all parameters in the software), click on the Excel® button at the bottom of the main window (indicated by the arrow on Figure 2).
Figure 2: ProSight Lite main window: the arrow indicates the button that exports the MS/MS data required by TDFragMapper.
188.8.131.52. Thermo® FreeStyleTM software (Xtract algorithm) : For deconvolving a MS/MS spectrum, follow the procedure as given in the Chapter 10 Deconvolving and Deisotoping Spectra with the Xtract Algorithm of the FreeStyle 1.4 user guide. Once the MS/MS spectrum has been deconvoluted, generate an output file as described below:
1. Select the Xtract Results tab
2. Click on the Workspace Options toolbar
3. Click on Selection As in the Exports tab (Figure 3)
4. Choose To CSV File as Export Type, click on OK
5. Save the *.xls output file
Figure 3: Thermo® FreeStyleTM software: the arrow indicates the button that exports the deconvoluted MS/MS data required by TDFragMapper.
2.2.4. To load all files set up in 2.2.3, click on the Next step button. The Filter tab will be enabled.
2.2.5. Creating Maps
184.108.40.206. To create a study map, three fixed conditions and one study condition need to be set up. The conditions* can be selected by using the associated drop-down menu (Figure 4).
*Available options: Fragmentation Method, Activation Level, Replicates and Precursor Charge State.
220.127.116.11.1. For each selected condition, all associated values found in the input tab are displayed in the left list. Desired values* can be selected or removed using the arrow buttons. TDFragMapper will apply default colors to the selected values of the study condition. To change the colors, double click on the selected value, choose a new color and validate with the OK button.
*For Fragmentation Method as first fixed condition, only one value is allowed to be selected
Figure 4: Example of a map for the purpose of studying the influence of the precursor charge states in HCD.
18.104.22.168.2. Add golden complementary pairs: This option allows the user to display golden complementary pairs on the final result image. A golden complementary pair is a pair of fragment ions (a/x, b/y or c/z) that have been formed by cleavage between the same pair of amino acids (Figure 5). The sum of the two fragments equals the mass of the targeted protein molecular ion , 
Figure 5: Golden complementary pairs generated in tandem mass spectrometry of intact proteins.
Golden complementary pairs will be represented as a star (*) under the corresponding inter-residue cleavage (Figure 7).
This option is allowed for maps with only 1 selected value per fixed condition
22.214.171.124.3. Add bond cleavage confidence: this option adds a color scale onto the protein sequence to highlight confidence in each proteolytic bond cleavage. The greater the number of matched fragment ions the darker the amino acid color (Figure 8).
126.96.36.199. One or more maps can be created. To create a new map, click on the Add another map button and repeat the 188.8.131.52 steps.
184.108.40.206. To display the fragmentation maps of the intact protein, click on the Display button. A new window will open.
3. Evaluating results
3.1. The fragmentation map of the intact protein is shown in the Display tab (Figure 6).
3.1.1. Fragments are represented as bars above (for N-terminal fragments) and below the protein sequence (for C-terminal fragments) and are split into panels according to their type (a-, b-, c-, x-, y- or z-ions). Fragments are organized in lines and are colored according to the values of the study condition (Figures 6-8).
3.1.2. To ease the evaluation of the resulting fragmentation map, a legend table is printed below each map and contains information on:
· the values of the study condition and the color code used,
· the percentage of residue cleavages,
· the number of matching fragments,
· the optional number of golden complementary pairs,
· the color scale of the optional bond cleavage confidence.
Figure 6: An example of a fragmentation map for the purpose of studying the influence of the precursor charge state in HCD with 20% activation.
Figure 7: An example of a fragmentation map for the purpose of studying the influence of the precursor charge state in HCD with 20% activation and with the golden complementary pair option enabled.
Figure 8: An example of a fragmentation map for the purpose of studying the influence of the precursor charge state in HCD with 20% activation and with the bond cleavage confidence option enabled.
3.2. Option Intensity: If the deconvoluted spectra files have been loaded (step 220.127.116.11), the Option Intensity tab is enabled, and information about the intensity of deconvoluted fragment ions can be added onto the fragmentation map.
3.2.1. Two options are available (Figure 9):
· Relative intensity: the relative abundance of deconvoluted fragment ions will be used (Figure 10).
· Absolute intensity: the sum intensity of deconvoluted fragment ions will be used (Figure 11)
3.2.2. After choosing one option, click on the Display button to evaluate the results. The visualization of ion intensity can be simply removed by unticking the “Per study map” button in the Option Intensity tab.
Figure 9: Option Intensity tab.
Figure 10: An example of a fragmentation map for the purpose of studying the influence of the precursor charge states in HCD with 20% activation considering the relative intensity of deconvoluted fragment ions.
Figure 11: An example of a fragmentation map for the purpose of studying the influence of the precursor charge states in HCD with 20% activation considering the absolute intensity of deconvoluted fragment ions.
3.3. Option Merging: the ultimate goal of TDFragMapper is to generate a final fragmentation map of an intact protein by combining all the fragments from the best MS/MS conditions
3.3.1 After having displayed and evaluated individual fragmentation maps, the user has the possibility to merge some conditions, through the Option Merging tab as shown in Figure 12.
18.104.22.168. Cleavage frequency: this option computes the frequency at which cleavages were find across the merged conditions and applies a corresponding color scale to the cleavages.
Figure 12: Option Merging – an example of setting up the combination of different studies previously created in different maps.
3.3.2. This final merged fragmentation map represents only N-Terminal cleavages (independently of the ion type a-, b- or c-) above the protein sequence and C-Terminal cleavages (independently of the ion type x-, y- or z-) below the protein sequence. The final percentage of residue cleavages is computed and displayed below the fragmentation map. If the Cleavage frequency option is enabled, cleavages will then be colored and the color scale will be displayed below the fragmentation map as can be seen in Figure 13.
Figure 13: Option Merging – an example of merged fragmentation maps with (top panel) and without (bottom panel) the Cleavage frequency option enabled.
3.4. Saving & exporting results
3.4.1. TDFragMapper allows to export the maps as image in the following formats: TIFF, PNG, JPG. This is done by clicking on File menu → Export → Image (or press CTRL + I), as shown in Figure 14.
3.4.2. TDFragMapper also allows to save the results which could later loaded back. This is done by clicking on File menu → Save Results (or press CTRL + S), as shown in Figure 14.
3.4.3. TDFragMapper also allows to export a summary report to PDF® file containing uploaded data files information and all the parameters used to create the maps. This is done by clicking on File menu → Export → Summary report (or by pressing CTRL + P), as shown in Figure 14.
Figure 14: Save results
3.5. Loading results
3.5.1. TDFragMapper loads results in its own format (*.tdfm). This can be accomplished in two ways, by double-clicking on a TDFragMapper results file or by clicking on File menu → Load Results (or press CTRL + O) and selecting the file, as seen in Figure 15.
Figure 15: Load TDFragMapper results from the main interface.
This tutorial can be accessed through Help menu → Read Me (or by pressing F1).