The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Abstract

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

The rise of the nanomaterial metabolite corona, and emergence of the complete corona

“…It is also pertinent to point out that multi-omic sample preparation techniques are increasing in popularity in recent years with a number of studies developing and validating methods that isolate both the proteins and metabolites of a given sample in similar ways as described above. [97][98][99][100] While it is likely that slight modifications may need to be made to these approaches to ensure the NMs themselves are also isolated to avoid injection into the analytical platforms, this is unlikely to be a major roadblock to the implementation to such approaches. Once the samples are prepared it is vital that they are correctly stored in order to prevent metabolite degradation.…”

“…Once the metabolite phases have been removed for analysis this would leave a protein layer which could then be digested and analysed for the protein corona. While similar approaches have been used in proteomics and metabolomics analyses from the same sample [97][98][99] this would require significant work to validate to ensure that the NMs are not mixed in with either biphase or the protein layer. As such the Bligh and Dyer approach may be the optimal starting point as the proteins are suspended between the two immiscible solvents with the NMs potentially sedimenting following the centrifugation step.…”

Soil Metaproteomics for the Study of the Relationships Between Microorganisms and Plants: A Review of Extraction Protocols and Ecological Insights

“…The method is based on the co-extraction of DNA, RNA, and proteins [ 80 ], which provides for the use of phenol-chloroform, which has been taken up and adapted in various works to allow the co-extraction from soil samples with sufficient yields to allow downstream analysis [ 81 , 82 , 83 ]. Given the potential of the multiomic approach for soil samples, to reduce time and costs, Nicora et al [ 84 ] proposed a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) protocol [ 84 ]. The protocol, previously used only for lipid extraction, involves the use of organic solvents, chloroform, methanol, and water.…”

“…Chloroform is not miscible with water and allows the three-phase chemical separation of the components: the aqueous phase contains the hydrophilic metabolites, the interface the proteins, and in the lower chloroform phase the lipid layer. The MPLEx protocol can be used on most soils; however, in highly organic soils such as peat, soil debris remains in the intermediate layer, negatively affecting protein extraction [ 84 ].…”

Uncovering hidden members and functions of the soil microbiome using de novo metaproteomics

“…All beads had previously been washed with chloroform and methanol and dried in a fume hood. Protein extraction occurred using a modified method of the Folch extraction 38 specifically for soil called Soil MPLex (Metabolite, protein, lipid extraction) 39 . Here, 4 mL of ice-cold ultrapure "Type 1" water (Millipore, Billerica, MA) was added to each sample and transferred to an ice bucket in a fume hood.…”