pTop 1.0: A High-Accuracy and High-Efficiency Search Engine for Intact Protein Identification

Abstract

There has been tremendous progress in top-down proteomics (TDP) in the past 5 years, particularly in intact protein separation and high-resolution mass spectrometry. However, bioinformatics to deal with large-scale mass spectra has lagged behind, in both algorithmic research and software development. In this study, we developed pTop 1.0, a novel software tool to significantly improve the accuracy and efficiency of mass spectral data analysis in TDP. The precursor mass offers crucial clues to infer the potential post-translational modifications co-occurring on the protein, the reliability of which relies heavily on its mass accuracy. Concentrating on detecting the precursors more accurately, a machine-learning model incorporating a variety of spectral features was trained online in pTop via a support vector machine (SVM). pTop employs the sequence tags extracted from the MS/MS spectra and a dynamic programming algorithm to accelerate the search speed, especially for those spectra with multiple post-translational modifications. We tested pTop on three publicly available data sets and compared it with ProSight and MS-Align+ in terms of its recall, precision, running time, and so on. The results showed that pTop can, in general, outperform ProSight and MS-Align+. pTop recalled 22% more correct precursors, although it exported 30% fewer precursors than Xtract (in ProSight) from a human histone data set. The running speed of pTop was about 1 to 2 orders of magnitude faster than that of MS-Align+. This algorithmic advancement in pTop, including both accuracy and speed, will inspire the development of other similar software to analyze the mass spectra from the entire proteins.

In situ mass spectrometry analysis of intact proteins and protein complexes from biological substrates

“…Top-down MS/MS must also consider labile PTMs and bound ligands (under native conditions). Tools designed for top-down MS/MS interpretation and database searching include ProSightPC [89] (now marketed by Thermo Scientific), TopPIC [90], SpectroGene [91], MSPathFinder [92], pTop [93] and TopMG [94]. ProsightPC has been integrated with Proteome Discoverer (Thermo Scientific) and considering the most recent Orbitrap MS instrumental developments bring improvements to intact protein analysis, indicates substantial commercial interest in developing top-down MS/MS into a routine procedure.…”

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

“…• 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).…”

“…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 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.…”

A perspective view of top‐down proteomics in snake venom research

“…The information (MS and MS/MS) obtained by analyzing intact proteins in TD proteomics is extremely complex, due to the overlap of multiple charge states, and cannot be interpreted with existing state‐of‐art BU software tools. Data analysis includes isotopic peak picking and deconvolution (e.g., TRASH, MS‐Deconv, Xtract and Decon2LS common algorithms), followed by database search of intact proteins from tandem mass spectra (e.g., MS‐Align+, ProSightPC, PIITA, ProteinGoggle, MS‐Align‐E, pTop and Big Mascot), and finally statistical validation of protein identification and characterization ( p ‐value, E ‐value and C ‐score). A number of software packages for analysis of TD proteomics data have been introduced (http://www.topdownproteomics.org).…”