Towards the characterization of the hidden world of small proteins in Staphylococcus aureus, a proteogenomics approach.
Stephan Fuchs, Martin Kucklick, Erik Lehmann, Alexander Beckmann, Maya Wilkens, Baban Kolte, Ayten Mustafayeva, Tobias Ludwig, Maurice Diwo, Josef Wissing, Lothar Jänsch, Christian H Ahrens, Zoya Ignatova, Susanne Engelmann
Author Information
Stephan Fuchs: Robert Koch Institute, Methodenentwicklung und Forschungsinfrastruktur (MF), Berlin, Germany.
Martin Kucklick: University of Technical Sciences Braunschweig, Institute for Microbiology, Braunschweig, Germany. ORCID
Erik Lehmann: University of Technical Sciences Braunschweig, Institute for Microbiology, Braunschweig, Germany. ORCID
Alexander Beckmann: University of Technical Sciences Braunschweig, Institute for Microbiology, Braunschweig, Germany. ORCID
Maya Wilkens: Robert Koch Institute, Methodenentwicklung und Forschungsinfrastruktur (MF), Berlin, Germany. ORCID
Baban Kolte: University of Hamburg, Institute of Biochemistry and Molecular Biology, Hamburg, Germany.
Ayten Mustafayeva: University of Technical Sciences Braunschweig, Institute for Microbiology, Braunschweig, Germany.
Tobias Ludwig: University of Technical Sciences Braunschweig, Institute for Microbiology, Braunschweig, Germany.
Maurice Diwo: University of Technical Sciences Braunschweig, Institute for Microbiology, Braunschweig, Germany. ORCID
Josef Wissing: Helmholtz Center for Infection Research GmbH, Cellular Proteomics, Braunschweig, Germany.
Lothar Jänsch: Helmholtz Center for Infection Research GmbH, Cellular Proteomics, Braunschweig, Germany.
Christian H Ahrens: Agroscope, Research Group Molecular Diagnostics, Genomics and Bioinformatics & SIB Swiss Institute of Bioinformatics, Basel, Switzerland. ORCID
Zoya Ignatova: University of Hamburg, Institute of Biochemistry and Molecular Biology, Hamburg, Germany. ORCID
Susanne Engelmann: University of Technical Sciences Braunschweig, Institute for Microbiology, Braunschweig, Germany. ORCID
Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 proteins with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids.
