De Novo Sequencing of Peptides from Top-Down Tandem Mass Spectra

Abstract

De novo sequencing of proteins and peptides is one of the most important problems in mass spectrometry-driven proteomics. A variety of methods have been developed to accomplish this task from a set of bottom-up tandem (MS/MS) mass spectra. However, a more recently emerged top-down technology, now gaining more and more popularity, opens new perspectives for protein analysis and characterization, implying a need for efficient algorithms to process this kind of MS/MS data. Here, we describe a method that allows for the retrieval, from a set of top-down MS/MS spectra, of long and accurate sequence fragments of the proteins contained in the sample. To this end, we outline a strategy for generating high-quality sequence tags from top-down spectra, and introduce the concept of a T-Bruijn graph by adapting to the case of tags the notion of an A-Bruijn graph widely used in genomics. The output of the proposed approach represents the set of amino acid strings spelled out by optimal paths in the connected components of a T-Bruijn graph. We illustrate its performance on top-down data sets acquired from carbonic anhydrase 2 (CAH2) and the Fab region of alemtuzumab.

Top-down analysis of protein samples by de novo sequencing techniques

“…We used the top-down datasets for CAH2 and the light chain of alemtuzumab published in (Vyatkina et al, 2015) and available at http://bioinf.spbau.ru/en/twister. In brief, intact CAH2, and alemtuzumab digested with papain and reduced, were analyzed by a reversed-phase liquid chromatography (RPLC) system coupled online with a Thermo LTQ Orbitrap Elite and Velos, respectively.…”

“…The input for our method constitutes a list N of amino acid strings endowed with offsets, derived from the deisotoped and charge state deconvoluted, often referred to simply as deconvoluted, MS/MS spectra as explained in (Vyatkina et al, 2015). In brief, the input spectra are first preprocessed, which comprises merging nearby peaks (i. e. the ones with approximately the same masses deconvoluted from different charge states), optional peak reflection followed by merging nearby peaks (i. e. the ones due to pairs of complementary fragment ions in the original spectra), and optional water-loss ion elimination.…”

“…Yet to the best of our knowledge, only three published algorithms make any use of top-down data to achieve the above-mentioned goal, and namely, the one from (Horn et al, 2000), which, however, did not result in a publicly available tool, TBNovo (Liu et al, 2014) that processes combined MS/MS datasets and utilizes top-down spectra as a scaffold for assembling bottom-up spectra, and our recently developed approach called Twister (Vyatkina et al, 2015). At the same time, primarily due to increasing accessibility of hybrid Orbitrap Fourier transform mass spectrometers, the top-down technology is nowadays rapidly gaining popularity, thus enforcing the need in efficient approaches to de novo peptide and protein sequencing from top-down spectra alone.…”

“…This work represents a continuation of our research summarized in (Vyatkina et al, 2015), where we introduced a method that extracts from deconvoluted MS/MS spectra a number of accurate sequence fragments of the proteins contained in a sample. Here we describe an algorithm that takes as input the fragments obtained this way, and combines them to produce a set of so-called aggregated strings, each endowed with direct and reversed offsets intended to match the masses of the two terminal fragments preceding and following it in the underlying protein sequence.…”

“…The proposed approach is implemented in a software tool Twister available at http://bioinf.spbau.ru/en/twister, which also incorporates the method introduced in (Vyatkina et al, 2015), and thus, takes as input a set of deconvoluted MS/MS spectra.…”