# Using ProteoCombiner to integrate bottom-up and top-down proteomics data to improve proteoform identification

Diogo Borges Lima, Mathieu Dupré, Magalie Duchateau, Quentin Giai Gianetto, Martial Jean-Pierre Rey, Mariette I Matondo, Julia Chamot-Rooke

► To cite this version:

Diogo Borges Lima, Mathieu Dupré, Magalie Duchateau, Quentin Giai Gianetto, Martial Jean-Pierre Rey, et al.. Using ProteoCombiner to integrate bottom-up and top-down proteomics data to improve proteoform identification. 2021. pasteur-03243560

HAL Id: pasteur-03243560

https://pasteur.hal.science/pasteur-03243560

Preprint submitted on 5 Nov 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Using ProteoCombiner to integrate bottom-up and top-down proteomics data to improve proteoform identification

**Diogo Borges Lima** (✉ diogobor@gmail.com)

Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France
https://orcid.org/0000-0001-6056-0825

**Mathieu Dupré**

Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France
https://orcid.org/0000-0002-1845-0048

**Magalie Duchateau**

Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France

**Quentin Giai Gianetto**

Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France

**Martial Rey**

Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France

**Mariette Matondo**

Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France

**Julia Chamot-Rooke** (✉ julia.chamot-rooke@pasteur.fr)

Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France

## Method Article

**Keywords:** proteomics, bioinformatics, top-down, bottom-up, mass spectrometry

**Posted Date:** February 17th, 2020

**DOI:** https://doi.org/10.21203/rs.2.10327/v1

**License:** © 🌐 This work is licensed under a Creative Commons Attribution 4.0 International License.

Read Full License
**Abstract**

Here we present a high-performance software for proteome analysis that combines different mass spectrometric approaches, such as, top-down for intact protein analyses and bottom-up, for proteolytic fragment characterization. **ProteoCombiner** capitalizes on the data arising from different experiments and proteomics search engines and presents the results in a user-friendly manner. Our tool also provides a rapid and easy visualization, manual validation and comparison of the identified proteoform sequences, including post-translational modifications (PTM) characterization. Thus, ProteoCombiner is recommended for studies dealing with different proteomics strategies, in order to increase confidence in proteoform identification including PTMs.

**Introduction**

Proteoforms describe all combinatorial sources of variation from a single gene, including genetic variations, alternative splicing, and post-translational modifications [1]. Proteoform analysis is nowadays of crucial importance since they have been proven to have a key role in biological systems [2] [3]. This includes both precise determination of the expressed sequence of the protein and the characterization of all Post Translational Modifications (PTM), in particular their precise localization and determination of their combination (combinatorial PTM). Several proteomics strategies, including bottom-up, middle-down and top-down proteomics, have been developed to analyze the PTM at the peptide level or to directly target proteoforms. Nevertheless, there is a lack of bioinformatics tools able to combine such proteomics results and it is still difficult to map the PTMs identified in bottom-up data onto proteoforms obtained by top-down proteomics.

In this protocol, we describe the key steps for using **ProteoCombiner**, a powerful tool capable of integrating proteomics datasets obtained from different experiments (bottom-up and top-down proteomics) and different search engines, such as, PatternLab for Proteomics[4], MaxQuant[5], Comet[6], ProsightPD[7], pTop[8] and TopPIC[9], in order to increase the confidence of proteoform identification and facilitate the characterization of PTMs. A video demonstrating ProteoCombiner v1.0 in action is available at https://proteocombiner.pasteur.fr/video.

**Reagents**

**Equipment**

**Hardware**

* A computer with at least 8 GB RAM and 2 computing cores is recommended.

**Software**
* Windows 10 (32 or 64 bits) or later.

* The .NET framework 4.7, which will be automatically updated by ProteoCombiner if necessary.

* ProteoCombiner software is available at https://proteocombiner.pasteur.fr.

## Data files

* **ProteoCombiner** v1.0 is compatible with output data files from PatternLab for Proteomics[4], MaxQuant[5], Comet[6], ProsightPD[7], pTop[8] and TopPIC[9] and can work directly with Thermo®, ABSciex®, Agilent® and Waters® RAW files. The software is also compatible with mzML 1.1.0, MS2 and Mascot Generic Format (MGF).

* **ProteoCombiner** saves results in its own format (i.e., *.pcmb) and exports them as Excel® (*.xlsx) and PDF® format files.

## Procedure

### 1. Software installation:

Download **ProteoCombiner** by clicking on the *Download* button at https://proteocombiner.pasteur.fr.

### 2. Workflow

The following workflow demonstrates how to combine proteomics data using **ProteoCombiner**.

#### 2.1. Execute the ProteoCombiner tool (Figure 1)

**[Figure 1]**

2.2. Specify the directory containing all result files from bottom-up proteomics experiments. In this directory can also have the database used in the search and the original RAW files in one of these formats: mzML 1.1.0, MS2, Mascot Generic Format (MGF), ABSciex®, Agilent®, Waters® and Thermo® RAW.

#### 2.2.1. Bottom-up proteomics
**2.4.1. Remove Contaminants:** This option allows to remove all protein sequences that represent a contaminant.

**2.4.2. Remove Reverse Sequence:** This option allows to remove all decoy protein sequences.

**2.5.** To start combining data, click on the OK button in the Combine tab.

PS: Although ProteoCombiner capitalizes on the data arising from different proteomics search engines, we recommend using a single software-tool to analyze all experiments for proteolytic fragment characterization, and another to analyze intact proteins experiments.

# 3. Exploring the results

Note: At this point we recommend saving results by selecting Save from File menu or by pressing CTRL + S.

## 3.1. Filter

**3.1.1.** All results are pre-filtered according to the following parameters designated on the top of the Results Browser window, as shown in **Figure 2**:

**3.1.1.1. CombScore:** Results containing identification scores greater than or equal to this value will be displayed.

**3.1.1.2. Peptide Count:** Only identified proteins containing at least this value as identified peptide amount (by bottom-up) will be displayed.

**3.1.1.3. Spectral Count:** Only identified proteins containing at least this value as identified spectra amount will be displayed.

**3.1.1.4. Unique peptide:** Only identified proteins containing at least this value as identified unique peptide amount will be displayed.

**3.1.1.5. Search:** Only results from peptides or proteins containing the sequence input to this field will be displayed. The user can further search by ProteinID (protein accession number), protein description or file name.

**3.1.1.6.** By clicking on *Filter* button, this operation is accomplished using all of the parameters described above.
**3.1.1.7.** By clicking on *Reset* button, all initial values are restored.

[Figure 2]

**3.2. Combined results**

All identified proteins that contain a valid sequence* will be displayed on this tab sorted by *Score* followed by *Sequence Coverage*. The protein score is represented by the best proteoform score.

*Protein sequence present in the database.*

**3.2.1.** By clicking on an identified protein, all respective identified proteoforms will be displayed* below the protein table sorted by *CombScore**\*\*.

*If there is no identified proteoform for a respective protein, all possible identified peptides will be displayed instead.*

**\*\*CombScore is calculated by summing two different scores: i) a score related to the TDP identification software (which is normalized between 0 and 1; and ii) the percentage of the proteoform sequence coverage based on the peptides, obtained in BUP approach, that match to this proteoform. This score also ranges between 0 and 1.**

**3.2.1.1.** The user can assess each identified proteoform by clicking on the *Is valid* checkbox.

**3.2.1.1.1.** At this point, we recommend saving results once again so that the personal assessments can be included. This is done by selecting *Save* from *File* menu or by pressing CTRL + S.

**3.2.1.2.** By clicking on an identified proteoform, all respective identified peptides will be displayed below the proteoform table sorted by *Peptide Score*.

**3.2.1.2.1** By double-clicking on an identified peptide, the tandem mass spectrum, which contains the best identification score, will be displayed on *Spectrum Viewer*. (**Figure 3**)

[Figure 3]

**3.2.2.** By double-clicking on an identified protein, a new window will be opened that shows the *Protein Coverage* (**section 3.5**).
### 3.3. Bottom-up proteomics results

All identified proteins and the corresponding peptides will be displayed on this tab.

**3.3.1.** By clicking on an identified protein, all respective identified peptides will be displayed below the protein table.

**3.3.1.1.** By clicking on an identified peptide, all respective identified tandem mass spectra will be displayed below the peptide table.

**3.3.1.1.1.** By double-clicking on an identified tandem mass spectrum, the *Spectrum Viewer* will be opened. (Figure 3)

**3.3.2.** By double-clicking on an identified protein, a new window will be opened that shows the *Protein Coverage* (**section 3.5**).

### 3.4. Top-down proteomics results

All identified proteoforms will be displayed on this tab grouped by *Theoretical Mass* in Da.

**3.4.1.** By double-clicking on the *Scan Number* column of an identified proteoform, the *Spectrum Viewer* will be opened. (Figure 3). By clicking on any other column, a new window will be opened that shows the *Protein Coverage* (**section 3.5**).

### 3.5. Protein Coverage

**3.5.1.** Once the window is opened, the information will be displayed on the top tab: Protein description, Monoisotopic and Average protein mass* and Sequence coverage. (Figure 4)

*If Protein coverage window is opened from the click of a specific proteoform, its monoisotopic and average mass will be displayed instead of the protein mass.*

[Figure 4]

**3.5.2.** The left box will display all approaches used to identify the proteoforms and/or peptides. By clicking on each item, all respective lines will be high-lighted in the right box.

**3.5.2.1.** Bottom-up and middle-down approaches, represented by the blue color,
**3.5.2.2.** Top-down approach is represented in three different colors: Expected proteoforms in orange, which means all identified proteoforms by the full theoretical mass; non-expected proteoforms in cyan, which represents all identified truncated proteoforms; and tagged proteoforms in red, which means all proteoforms that were identified by a part of the protein sequence.

**3.5.2.3.** All identified PTMs will also be displayed in this box.

**3.5.3.** The right box will display all identified proteoforms and/or peptides that will be represented by different lines. All of them will be displayed sorted by *CombScore*. On the top will be displayed the full protein sequence and all theoretical modifications (present in the database) as can be seen in **Figure 4**. All theoretical chains will be shown below protein sequence in gray dash lines. The user is able to check all information about the modification or theoretical chain by passing the mouse over the line or the modified amino acid. (Figure 4)

**3.5.3.1.** Each proteoform will be displayed according to the classification: expected, non-expected or tagged proteoform. By right-clicking on each line, the user is able to assess the proteoform identification (valid or invalid); in addition, it's possible to highlight only the peptides that fit into the proteoform. (Figure 5). By hovering over each line, some information will be shown: Proteoform sequence, Score, Search engine that identified this specific sequence, Start and End positions.

**3.5.3.2.** All identified PTMs will also be displayed and by hovering over each one, it is possible to check their position and description.

[Figure 5]

**3.5.3.3.** The identified proteoforms and/or peptides can be displayed in a single line or in multiple lines. The user can change the way to visualize in *Utils* menu, by selecting *Custom Protein Visualization* option in *Results Browser* window (or by pressing ALT + C).

**3.6. Loading results**

**3.6.1. ProteoCombiner** loads results in its own format (\*.pcmb). This can be accomplished in three ways, the easiest one is by double-clicking on a ProteoCombiner results file. If the *Results Browser* window is opened, another way to launch the file is by clicking on *Load* option from the *File* menu or by pressing CTRL + O, as seen in (Figure 6). Otherwise, if the main window is opened, select *Load Results* from *File* menu (or press CTRL + O), as seen in Figure 7.

[Figure 6]

[Figure 7]
### 3.7. Exporting results

**3.7.1. ProteoCombiner** also allows to export all combined results to Excel® (*.xlsx) or PDF® file. This is done by selecting *Excel file* from the File menu ▷ Export results (or by pressing ALT + E). Or by selecting *PDF file* from the File menu ▷ Export results (or by pressing ALT + P). **(Figure 6)**

## Troubleshooting

### Time Taken

### Anticipated Results

### References

[1] L. M. Smith and N. L. Kelleher, "Proteoforms as the next proteomics currency," *Science*, vol. 359, no. 6380, pp. 1106-1107, Mar. 2018.

[2] Z. R. Gregorich and Y. Ge, "Top-down proteomics in health and disease: Challenges and opportunities," *PROTEOMICS*, vol. 14, no. 10, pp. 1195-1210, 2014.

[3] J. Chamot-Rooke et al., "Posttranslational Modification of Pili upon Cell Contact Triggers N. meningitidis Dissemination," *Science*, vol. 331, no. 6018, pp. 778-782, Feb. 2011.

[4] P. C. Carvalho et al., "Integrated analysis of shotgun proteomic data with PatternLab for proteomics 4.0," *Nat. Protoc.*, vol. 11, no. 1, pp. 102-117, Dec. 2015.

[5] J. Cox, N. Neuhauser, A. Michalski, R. A. Scheltema, J. V. Olsen, and M. Mann, "Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment," *J. Proteome Res.*, vol. 10, no. 4, pp. 1794-1805, Apr. 2011.

[6] J. K. Eng, T. A. Jahan, and M. R. Hoopmann, "Comet: an open-source MS/MS sequence database search tool," *Proteomics*, vol. 13, no. 1, pp. 22-24, Jan. 2013.

[7] "ProSightPD 1.1: Integrating Top Down Searching in Proteome Discoverer 2.1.".

[8] R.-X. Sun et al., "pTop 1.0: A High-Accuracy and High-Efficiency Search Engine for Intact Protein Identification," *Anal. Chem.*, vol. 88, no. 6, pp. 3082-3090, Mar. 2016.

[9] Q. Kou, L. Xun, and X. Liu, "TopPIC: a software tool for top-down mass spectrometry-based proteoform identification and characterization," *Bioinforma. Oxf. Engl.*, vol. 32, no. 22, pp. 3495-3497, Nov. 2016.
# Acknowledgements

The authors thank the Agence Nationale de la Recherche (project ANR-15-CE18-0021) for financial support. This work is also part of the European Joint Programme One Health EJP from the European Union's Horizon 2020 research and innovation programme (Grant Agreement 773830).

# Figures

<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

Graphical User Interface of the main window of ProteoCombiner.

<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

Filter parameters responsible for selecting results.
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>

Spectrum viewer – Blue lines represent all y or x or z peaks matched, whereas red lines represent all a or b or c peaks matched. Yellow lines represent the precursor ion.

<figure><img src="image_5.png" /><figcaption>Figure 4</figcaption></figure>

Protein coverage window: All identified proteoforms and/or peptides are displayed here in different lines. The theoretical sequences (proteoforms) are displayed in gray dash lines and the theoretical PTMs above
protein sequence. The information about the PTM and theoretical sequences are shown when the mouse is hovered over them.

<figure><img src="image_6.png" /><figcaption>Figure 5</figcaption></figure>

An assessment can be applied for each identified proteoform in order to increase the confidence of the evaluation. The user can also highlight only peptides that fit with a specific proteoform.

<figure><img src="image_7.png" /><figcaption>Figure 6</figcaption></figure>

Save or Load ProteoCombiner results from Results Browser window. The user can also export the results as an Excel® or PDF format file.
<figure><img src="image_8.png" /><figcaption>Figure 7</figcaption></figure>

Load ProteoCombiner results from main interface.