ProteoFormer - a software toolkit for top-down proteomics

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Most proteomics workflows employ a "bottom-up" method, in which the identity and intensity of peptide ions measured by mass spectrometry (MS) are used as a proxy measure for the parent protein, since signals from analysis of intact proteins were too complex to interpret on older, low-resolution instruments. Recent improvements mean that the isotope pattern of large molecular weight species can now be resolved, enabling direct analysis of intact proteins - "top-down" proteomics. A major advantage of top-down analysis is the ability to characterise closely related proteins (proteoforms), resulting from paralogs, alternative splicing or different post-translational modifications (PTMs) on proteins.

MS data from intact proteins can be hugely complex to interpret, and the majority of software tools have been developed for peptide data. In this project, we will develop software to tackle two related problems. Firstly, ionisation of intact proteins generates high-charge state ions, often with overlapping signals. We have previously developed the seaMass software, which is able to identify, separate and de-charge overlapping isotope patterns in complex LC-MS peptide data robustly under ion counting noise. We will adapt seaMass for detecting high-charge state, intact protein data, producing de-charged MS1 and MS2 spectra. Secondly, the traditional sequence database search used in peptide identification (in which modifications must be specified in advance) is not suitable for intact protein data. We are developing the Proteoformer software, which converts a fragment spectrum into a set of peptide regular expressions (pep regexes) that are used to search a protein database index. Following identification of the correct database protein, we can dynamically identify the PTMs and processing events that have occurred de novo. Both software packages will be provided with a graphical interface through our Proteosuite software, and will work with open data standards.

Our developments will have impacts through the following routes:

- The development of seaMass-TD and Proteoformer will make it more straightforward for top-down analysis to be performed on a much wider range of instruments, producing high-quality and reliable results. This will open up this important technology for studying proteins in their native state in the cell, for basic and applied research across numerous domains.

- Our software has the potential to increase sales of mass spectrometers, capable of performing top-down analysis. In particular, locally we are working with Waters to develop software compatible with their data, since current software has typically been designed for mass spectrometers produced by Thermo.

- We will explore routes for commercialisation of Proteoformer and seaMass-TD, as discussed in the Pathways to Impact document.

- We will work with international consortia aimed with data sharing and standardisation in proteomics - the Proteomics Standards Initiative (PSI), ProteomeXchange and EBI's PRIDE database to ensure that current standards can appropriately handle top-down data, and researchers can submit data to the leading public repositories for community re-analysis.