Original Research ARTICLE
Evaluation of sample preparation methods for fast proteotyping of microorganisms by tandem mass spectrometry
- 1CEA Marcoule, France
- 2Laboratoire Innovations technologiques pour la Détection et le Diagnostic (Li2D), Service de Pharmacologie et Immunoanalyse (SPI), Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), France
Tandem mass spectrometry-based proteotyping allows characterizing microorganisms in terms of taxonomy and is becoming an important tool for investigating microbial diversity from several ecosystems. Fast and automatable sample preparation for obtaining peptide pools amenable to tandem mass spectrometry is necessary for enabling proteotyping as a high-throughput method. First, the protocol to increase the yield of lysis of several representative bacterial and eukaryotic microorganisms was optimized by using a long and drastic bead-beating setting with 0.1mm silica beads, 0.1mm and 0.5mm glass beads, in presence of detergents. Then, three different methods to obtain greater digestion yield from these extracts were tested and optimized for improve efficiency and reduce application time: denaturing electrophoresis of proteins and in-gel proteolysis, suspension-trapping filter-based approach (S-Trap) and, solid-phase-enhanced sample preparation named SP3. The latter method outperforms the other two in terms of speed and delivers also more peptides and proteins than with the in-gel proteolysis (2.2 fold for both) and S-trap approaches (1.3 and 1.2 fold, respectively). Thus, SP3 directly improves tandem mass spectrometry proteotyping.
Keywords: detection, IDENTIFICATION, Mass Spectrometry, Proteotyping, Shotgun Proteomics, Microorganism, sample preparation
Received: 06 May 2019;
Accepted: 13 Aug 2019.
Edited by:Peter Lasch, Robert Koch Institute, Germany
Reviewed by:Céline Henry, INRA Centre Jouy-en-Josas, France
Robert Heyer, Otto von Guericke University Magdeburg, Germany
Copyright: © 2019 Hayoun, Gouveia, Grenga, Pible, ARMENGAUD and ALPHA-BAZIN. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Dr. Jean ARMENGAUD, Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Laboratoire Innovations technologiques pour la Détection et le Diagnostic (Li2D), Service de Pharmacologie et Immunoanalyse (SPI), Paris, F-30207, France, email@example.com