SDS‐PAGE fractionation to increase metaproteomic insight into the taxonomic and functional composition of microbial communities for biogas plant samples

Wenzel, Lisa; Heyer, Robert; Schallert, Kay; Löser, Lucy; Wünschiers, Röbbe; Reichl, Udo; Benndorf, Dirk

doi:10.1002/elsc.201800062

Cited by 8 publications

(6 citation statements)

References 33 publications

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“…The total number of protein groups and unique peptide identifications was comparable between the GeLC approach and our best performing 2D-LC-MS/MS approach (2D|10pH_G1) and thus higher than our 1D-LC-MS/MS-only approaches. A similar performance difference was recently observed in a study of biogas plant communities, where a GeLC method performed much better than a 120 min 1D-LC-MS/MS method (Wenzel et al, 2018). The GeLC method also performed comparably to the 2D methods in terms of run reproducibility (Figure 3) and identification of protein groups of low-abundance organisms (Figure 4).…”

Section: Resultssupporting

confidence: 79%

More Is Not Always Better: Evaluation of 1D and 2D-LC-MS/MS Methods for Metaproteomics

et al. 2019

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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.

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Section: Resultssupporting

confidence: 79%

More Is Not Always Better: Evaluation of 1D and 2D-LC-MS/MS Methods for Metaproteomics

et al. 2019

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“…The differences in acquired spectra show a clear relation to the method used, as similar methods or replicates show highly similar numbers of acquired spectra. As expected, more complex methods with longer gradient lengths, fractionation, and additional separation methods such as MudPIT 50 or ion mobility (PASEF) 51 led to up to eight times more identified spectra, but at the cost of increased time and resources spent 52 (see Supplementary Table 1 for a detailed description, and Supplementary Table 2 for an overview of the samples). Notably, identification rates were not necessarily correlated with the total number of identifications.…”

Section: Resultssupporting

confidence: 53%

“…Furthermore, each algorithm uses its own score as a quality metric for finding the best matching peptide for a spectrum. This score varies between the search engines and can even result in different peptide identifications for the same spectrum 54 .…”

Section: Different Bioinformatic Pipelines Resulted In Highly Similar Peptide Identificationsmentioning

confidence: 99%

Critical Assessment of Metaproteome Investigation (CAMPI): A Multi-Lab Comparison of Established Workflows

Bossche

Kunath

Schallert

et al. 2021

Preprint

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Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carried out the first community-driven, multi-lab comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluated the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, lab-assembled human intestinal model and a human fecal sample. We observed that variability at the peptide level was predominantly due to wet-lab workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappeared at protein group level. While differences were observed for predicted community composition, similar functional profiles were obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-lab studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

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“…Functional changes in the pathway abundances of low abundant SIHUMIx bacteria were not detected. This is most likely due to the low proteome coverage of low abundant species (less than 1%), which is still a bottleneck in metaproteome analysis [48,49].…”

Section: A Faster Transit Time Slightly Reduces Butyrate Metabolism Omentioning

confidence: 99%

The Simplified Human Intestinal Microbiota (SIHUMIx) Shows High Structural and Functional Resistance against Changing Transit Times in In Vitro Bioreactors

et al. 2019

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Many functions in host-microbiota interactions are potentially influenced by intestinal transit times, but little is known about the effects of altered transition times on the composition and functionality of gut microbiota. To analyze these effects, we cultivated the model community SIHUMIx in bioreactors in order to determine the effects of varying transit times (TT) on the community structure and function. After five days of continuous cultivation, we investigated the influence of different medium TT of 12 h, 24 h, and 48 h. For profiling the microbial community, we applied flow cytometric fingerprinting and revealed changes in the community structure of SIHUMIx during the change of TT, which were not associated with changes in species abundances. For pinpointing metabolic alterations, we applied metaproteomics and metabolomics and found, along with shortening the TT, a slight decrease in glycan biosynthesis, carbohydrate, and amino acid metabolism and, furthermore, a reduction in butyrate, methyl butyrate, isobutyrate, valerate, and isovalerate concentrations. Specifically, B. thetaiotaomicron was identified to be affected in terms of butyrate metabolism. However, communities could recover to the original state afterward. This study shows that SIHUMIx showed high structural stability when TT changed-even four-fold. Resistance values remained high, which suggests that TTs did not interfere with the structure of the community to a certain degree.

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SDS‐PAGE fractionation to increase metaproteomic insight into the taxonomic and functional composition of microbial communities for biogas plant samples

Cited by 8 publications

References 33 publications

More Is Not Always Better: Evaluation of 1D and 2D-LC-MS/MS Methods for Metaproteomics

More Is Not Always Better: Evaluation of 1D and 2D-LC-MS/MS Methods for Metaproteomics

Critical Assessment of Metaproteome Investigation (CAMPI): A Multi-Lab Comparison of Established Workflows

The Simplified Human Intestinal Microbiota (SIHUMIx) Shows High Structural and Functional Resistance against Changing Transit Times in In Vitro Bioreactors

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