Using TDFragMapper: a high-performance tool to assess experimental parameters in top-down proteomics

Here we present a new software-tool for visualizing fragment ions and sequence coverage of intact proteins in top-down mass spectrometry. TDFragMapper combines the data arising from multiple and diverse tandem mass spectrometry experiments of intact proteins. Our tool maps fragment ions onto the protein backbone sequence and allows for a rapid comparison of the results obtained when varying experimental parameters in tandem mass spectrometry (MS/MS) experiments of intact proteins: fragmentation method, selected precursor charge state, activation level, technical replicate. In summary, TDFragMapper is a unique software-tool that allows an easy selection of the best experimental conditions leading to the highest condence in intact protein sequence identication.


Introduction
Top-down proteomics (TDP) is a powerful technology allowing the characterization of proteins at the proteoform level using high-resolution tandem mass spectrometry (MS/MS). The complete characterization of proteoforms often requires the use of several complementary fragmentation techniques, such as collision-induced dissociation (CID), electron transfer dissociation (ETD) or ultraviolet photodissociation (UVPD) [1]. In contrast to bottom-up proteomics, the experimental parameters used for the fragmentation in TDP, such as the activation energy or the charge state of the precursor ion chosen for fragmenting an intact protein, can signi cantly affect the quality of MS/MS data and therefore the protein sequence coverage [2]. Although existing tools are capable of matching a list of fragment ions to a protein sequence (e.g., ProSight Lite [3]), there is currently no computational tool allowing to visualize fragments arising from diverse MS/MS experiments on a unique fragmentation map without losing information on the contribution and the speci city of each experiment. Current tools provide a unique fragmentation map per MS/MS experiment, and thus their comparison, in particular when multiple parameters are assessed, is both di cult and time-consuming. Moreover, although the intensity of fragment ions can represent a precious source of information when dealing with MS/MS data, it is often not considered when matching fragments onto a protein sequence. To circumvent these limitations, here we introduce TDFragMapper, a novel software-tool that can display and combine pre-assigned fragment ions achieved from various MS/MS experiments on a unique protein sequence, keeping an easy access to the individual contribution of each experiment and to the intensity of deconvoluted fragment ions. Our tool makes it possible to rapidly compare experimental parameters such as the type of fragmentation, the activation level or the precursor charge state in the MS/MS analysis of intact proteins.
In this protocol, we describe the key steps for using TDFragMapper, which enables to compare fragment ions arising from different tandem mass spectrometry experiments by taking into consideration the contribution of each experiment, the intensity of deconvoluted fragment ions and the presence of golden complementary pairs. Reagents Equipment Hardware •A computer with at least 8 GB RAM and 2 computing cores is recommended.
•The .NET framework 4.8, which will be automatically updated by TDFragMapper if necessary.
•TDFragMapper saves results in its own format (i.e., *.tdfm) and exports the analysis to an image in TIFF, PNG or JPG format.    (Figure 2). Specify a Deconvoluted Spectra le that was generated by Thermo® FreeStyle™ software (Xtract algorithm) [4] if an evaluation of fragment ions intensity is desired.

2.2.3.1.
To add new data le parameters, click on the Add another data le button. A new line will be included in the table.
2.2.3.1.1. Instead of lling le by le, the user has the possibility to ll multiple data les at once by copying from a table (e.g., Excel® le), and pasting by right-clicking on the table or pressing CTRL + V. The table (e.g., Excel® le) 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:      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.    3.3.2. This nal 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 nal 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.

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® le containing uploaded data les 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.    Example of a map for the purpose of studying the in uence of the precursor charge states in HCD.

Figure 5
Golden complementary pairs generated in tandem mass spectrometry of intact proteins      Save results

Figure 15
Load TDFragMapper results from the main interface. Figure 16