Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria

• Main Authors: Anna A. Toymentseva, Anastasia O. Koryagina, Alexander V. Laikov, Margarita R. Sharipova
• Format: Article
• Language: English
• Published: MDPI AG 2020-07-01
• Series: International Journal of Molecular Sciences
• Subjects: mass spectrometry; multiple reaction monitoring (MRM); protein quantification; subtilisin-like protease (AprBp); glutamyl endopeptidase (GseBp); <i>Bacillus pumilus</i>
• Online Access: https://www.mdpi.com/1422-0067/21/14/4924

<i>Bacillus subtilis</i> produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type <i>B. pumilus</i> cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant <i>B. subtilis</i> cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of <i>B. pumilus</i> strain in comparison to the recombinant <i>B. subtilis</i> strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.