Abstract
Metaproteomics, the study of protein expression in microbial communities, is a versatile tool for environmental microbiology. Achieving sufficiently high metaproteome coverage to obtain a comprehensive picture of the activities and interactions in microbial communities is one of the current challenges in metaproteomics. An essential step to maximize the number of identified proteins is peptide separation via liquid chromatography (LC) prior to mass spectrometry (MS). Thorough optimization and comparison of LC methods for metaproteomics are, however, currently lacking. Here, we present an extensive development and test of different 1D and 2D-LC approaches for metaproteomic peptide separations. We used fully characterized mock community samples to evaluate metaproteomic approaches with very long analytical columns (50 and 75 cm) and long gradients (up to 12 h). We assessed a total of over 20 different 1D and 2D-LC approaches in terms of number of protein groups and unique peptides identified, peptide spectrum matches (PSMs) generated, the ability to detect proteins of low-abundance species, the effect of technical replicate runs on protein identifications and method reproducibility. We show here that, while 1D-LC approaches are faster and easier to set up and lead to more identifications per minute of runtime, 2D-LC approaches allow for a higher overall number of identifications with up to >10,000 protein groups identified. We also compared the 1D and 2D-LC approaches to a standard GeLC workflow, in which proteins are pre-fractionated via gel electrophoresis. This method yielded results comparable to the 2D-LC approaches, however with the drawback of a much increased sample preparation time. Based on our results, we provide recommendations on how to choose the best LC approach for metaproteomics experiments, depending on the study aims.
Highlights
Metaproteomics, the analysis of expressed proteins in a microbial community (Wilmes and Bond, 2004), is a powerful tool which has enabled new insights into the role of microorganisms in a variety of environments
While 2D-liquid chromatography (LC) methods can lead to more total identifications, they are not necessarily the best choice for metaproteomic studies. 1D-LC methods with long columns and gradients offer a suitable alternative in terms of throughput and ease of use
Our system had separate strong cation exchange (SCX) and C18 pre-columns and a switching valve that connected the C18 pre-column to the analytical column, while Taylor et al (2009) used a biphasic pre-column connected to the analytical column in a vented-column setup
Summary
Metaproteomics, the analysis of expressed proteins in a microbial community (Wilmes and Bond, 2004), is a powerful tool which has enabled new insights into the role of microorganisms in a variety of environments. In the most common metaproteomic workflow, the protein mixture which is extracted from an environmental sample is digested into peptides. This peptide mixture is analyzed in a mass spectrometer. There are numerous challenges associated with this workflow that must be addressed in order to obtain the largest possible number of identified and quantified proteins. For two comprehensive reviews that provide more details on these challenges and how they can be addressed see VerBerkmoes et al (2009) and Wilmes et al (2015)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
