title: Navigating through metaproteomics data: a logbook of database searching.
abstract: Metaproteomic research involves various computational challenges during the identification of fragmentation spectra acquired from the proteome of a complex microbiome. These issues are manifold and range from the construction of customized sequence databases, the optimal setting of search parameters to limitations in the identification search algorithms themselves. In order to assess the importance of these individual factors, we studied the effect of strategies to combine different search algorithms, explored the influence of chosen database search settings, and investigated the impact of the size of the protein sequence database used for identification. Furthermore, we applied de novo sequencing as a complementary approach to classic database searching. All evaluations were performed on a human intestinal metaproteome dataset. Pyrococcus furiosus proteome data were used to contrast database searching of metaproteomic data to a classic proteomic experiment. Searching against subsets of metaproteome databases and the use of multiple search engines increased the number of identifications. The integration of P. furiosus sequences in a metaproteomic sequence database showcased the limitation of the target-decoy-controlled false discovery rate approach in combination with large sequence databases. The selection of varying search engine parameters and the application of de novo sequencing represented useful methods to increase the reliability of the results. Based on our findings, we provide recommendations for the data analysis that help researchers to establish or improve analysis workflows in metaproteomics.
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Muth
authors.affiliation: Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Carolin A
authors.initials: CA
authors.lastname: Kolmeder
authors.affiliation: Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Jarkko
authors.initials: J
authors.lastname: Salojärvi
authors.affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: Salla
authors.initials: S
authors.lastname: Keskitalo
authors.affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: Markku
authors.initials: M
authors.lastname: Varjosalo
authors.affiliation: Department of General Surgery, NUTRIM, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Froukje J
authors.initials: FJ
authors.lastname: Verdam
authors.affiliation: Department of General Surgery, NUTRIM, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Sander S
authors.initials: SS
authors.lastname: Rensen
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.|Otto-von-Guericke University, Bioprocess Engineering, Magdeburg, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Reichl
authors.affiliation: Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.|Department of Bacteriology and Immunology, University of Helsinki, Helsinki, Finland.|Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.
authors.forename: Willem M
authors.initials: WM
authors.lastname: de Vos
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.
authors.forename: Erdmann
authors.initials: E
authors.lastname: Rapp
authors.affiliation: Department of Biochemistry, Ghent University, Ghent, Belgium.|Department of Medical Protein Research, VIB, Ghent, Belgium.
authors.forename: Lennart
authors.initials: L
authors.lastname: Martens
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence / Q000235:genetics; D006801:Humans; D054892:Metagenome / Q000235:genetics*; D020543:Proteome / Q000235:genetics*; D040901:Proteomics; D020027:Pyrococcus furiosus / Q000235:genetics; D012984:Software; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome
keywords: Bioinformatics; De novo sequencing; False discovery rate; Metaproteomics; Search parameters