De Novo Protein Sequencing by Combining Top-Down and Bottom-Up Tandem Mass Spectra

Abstract

There are two approaches for de novo protein sequencing: Edman degradation and mass spectrometry (MS). Existing MS-based methods characterize a novel protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the protein. Because each tandem mass spectrum covers only a short peptide of the target protein, the key to high coverage protein sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resolution mass spectrometers have become accessible to many laboratories. These mass spectrometers are capable of analyzing molecules of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a protein. We propose an algorithm, TBNovo, for de novo protein sequencing by combining top-down and bottom-up MS. In TBNovo, a top-down tandem mass spectrum is utilized as a scaffold, and bottom-up tandem mass spectra are aligned to the scaffold to increase sequence coverage. Experiments on data sets of two proteins showed that TBNovo achieved high sequence coverage and high sequence accuracy.

De novo sequencing of proteins and peptides: algorithms, applications, perspectives

“…В 2014 г. был предложен комбинированный метод TBNovo для de novo секвенирования белков по наборам масс-спектров "сверху вниз" и "снизу вверх" [71], в соответствии с которым вначале выполнялось de novo секвенирование пептидов по данным "снизу вверх" с применением программы PEAKS, а затем полученные пептидные последовательности объединялись в белковую с использованием "каркаса" (scaffold), сформированного на основе данных "сверху вниз". Первым же алгоритмом de novo секвенирования белков лишь по масс-спектрам "сверху вниз", реализованным в виде свободно распространяемого программного инструмента, стал Twister [30][31][32].…”

Challenges and developments in protein identification using mass spectrometry

“…A further approach is the combination of complementary bottom-up and top-down MS/MS fragmentation [42,43], which aids the alignment of peptide fragments from a bottom-up analysis to the intact protein sequence.…”

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

“…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.…”