Abstract
Top-Down Proteomics (TDP) is an emerging proteomics protocol that involves identification, characterization, and quantitation of intact proteins using high-resolution mass spectrometry. TDP has an edge over other proteomics protocols in that it allows for: (i) accurate measurement of intact protein mass, (ii) high sequence coverage, and (iii) enhanced identification of post-translational modifications (PTMs). However, the complexity of TDP spectra poses a significant impediment to protein search and PTM characterization. Furthermore, limited software support is currently available in the form of search algorithms and pipelines. To address this need, we propose ‘SPECTRUM’, an open-architecture and open-source toolbox for TDP data analysis. Its salient features include: (i) MS2-based intact protein mass tuning, (ii) de novo peptide sequence tag analysis, (iii) propensity-driven PTM characterization, (iv) blind PTM search, (v) spectral comparison, (vi) identification of truncated proteins, (vii) multifactorial coefficient-weighted scoring, and (viii) intuitive graphical user interfaces to access the aforementioned functionalities and visualization of results. We have validated SPECTRUM using published datasets and benchmarked it against salient TDP tools. SPECTRUM provides significantly enhanced protein identification rates (91% to 177%) over its contemporaries. SPECTRUM has been implemented in MATLAB, and is freely available along with its source code and documentation at https://github.com/BIRL/SPECTRUM/.
Highlights
Top-Down Proteomics (TDP) is an emerging proteomics protocol that involves identification, characterization, and quantitation of intact proteins using high-resolution mass spectrometry
Elaborate graphical user interfaces (GUIs) have been provided for each step in the search process (Fig. 2b–f) and the summary of search results can be visualized as a ranked protein list (Fig. 2g)
We have proposed SPECTRUM, an open-source and open-architecture MATLAB toolbox for proteoform identification in top-down proteomics
Summary
Top-Down Proteomics (TDP) is an emerging proteomics protocol that involves identification, characterization, and quantitation of intact proteins using high-resolution mass spectrometry. Limited software support is currently available in the form of search algorithms and pipelines To address this need, we propose ‘SPECTRUM’, an open-architecture and open-source toolbox for TDP data analysis. Its salient features include: (i) MS2-based intact protein mass tuning, (ii) de novo peptide sequence tag analysis, (iii) propensity-driven PTM characterization, (iv) blind PTM search, (v) spectral comparison, (vi) identification of truncated proteins, (vii) multifactorial coefficient-weighted scoring, and (viii) intuitive graphical user interfaces to access the aforementioned functionalities and visualization of results. Reported MSPathFinder, a high-throughput tool employing parametric dynamic programming for spectral alignment, uses sequence graphs for efficient filtering of combinatorial proteoforms It lacks support for searching unknown modifications and its CLI makes it difficult to use. SPECTRUM results reported up to 47% more spectral matches and over 91% more proteins in comparison with other tools (see Case Study II – Results Section)
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