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

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.

Environmental Viscosity Modulates Interbacterial Killing during Habitat Transition

“…LC-MS/MS. All samples were analyzed by one-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously (70), with the modification that a 75-cm analytical column and a 140-min-long gradient were used. For each sample run, 400 ng peptide was loaded with an UltiMate 3000 RSLCnano liquid chromatograph (Thermo Fisher Scientific) in loading solvent A (2% acetonitrile, 0.05% trifluoroacetic acid) onto a 5-mm, 300-m (inside diameter) C 18 Acclaim PepMap100 precolumn (Thermo Fisher Scientific).…”

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

“…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].…”

A shared core microbiome in soda lakes separated by large distances

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