Highly Robust de Novo Full-Length Protein Sequencing

peer-reviewed · Analytical Chemistry · 2021

peer-reviewed · Analytical Chemistry · 2021. Zhi-Biao Mai et al. High Resolution Image Download MS PowerPoint Slide Accurate full-length sequencing of a purified unknown…
Date 2021-11-23
Type peer-reviewed
Venue Analytical Chemistry
Publisher ACS
Contribution algorithm
DOI 10.1021/acs.analchem.1c03718
Citations (OpenAlex) 22
Venue 2-year citedness 6.29

Abstract

High Resolution Image Download MS PowerPoint Slide Accurate full-length sequencing of a purified unknown protein is still challenging nowadays due to the error-prone mass-spectrometry (MS)-based methods. De novo identified peptide sequence largely contain errors, undermining the accuracy of assembly. Bias on the detectability of the peptides also makes low-coverage regions, resulting in gaps. Although recent advances on multi-enzyme hydrolysis and algorithms showed complete assembly of full-length protein sequences in a few examples, the robustness in practical application is still to be improved. Here, inspired by genome assembly strategies, we demonstrate a contig-scaffolding strategy to assemble protein sequences with high robustness and accuracy. This strategy integrates multiple unspecific hydrolysis methods to minimize the bias in the hydrolysis process. After de novo identification of the peptides, our assembly algorithm, named Multiple Contigs & Scaffolding (MuCS), assembles the peptide sequences in a multistep, i.e., contig-scaffold manner, with error correction in each step. MS data from different hydrolysis experiments complement each other for robust contig extension and error correction. We demonstrated that our strategy on three proteins and three replications all reached 100% coverage (except one with 98.85%) and 98.69–100% accuracy. It can also efficiently deal with the membrane protein, although the transmembrane region was missing due to the limitation of the MS. The three replicates reached 88.85–92.57% coverage and 97.57–100% accuracy. In sum, we provided a practical, robust, and accurate solution for full-length protein sequencing. The MuCS software is available at http://chi-biotech.com/mucs/ .

Authors

  1. Zhi-Biao Mai · Chinese Academy of Sciences, Jinan University
  2. Zhong-Hua Zhou · Jinan University
  3. Qing-Yu He · Jinan University
  4. Gong Zhang · Jinan University

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