MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses

Abstract

In systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample.

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

“…The A. thaliana MPLEx results were compared to an extraction performed with trichloroacetic acid (TCA) because plant leaves are rich in phenolic compounds that need to be removed prior to MS analyses 39 . In all cases, the number of proteins observed from the MPLEx method was similar to that for the controls 10 , indicating its utility for many different sample types. Also, there were no trends associated with the specific protein classes extracted in all the sample types (including soil).…”

“…The broad applicability of the MPLEx approach, has been previously evaluated using a diverse set of samples, including the archaeon Sulfolobus acidocaldarius , a unicyanobacterial consortium 38 , mouse brain cortex tissue, human urine, and leaves from Arabidopsis thaliana (Figure 2) 10 .…”

“…These precise procedures make their implementation extremely difficult or impossible when only a limited quantity of sample is available. These challenges have prompted us to investigate a simultaneous metabolite, protein, and lipid extraction (MPLEx) method capable of using smaller sample volumes or masses, improving accuracy, and providing faster sample preparations for all three analyses 10 . To date, there are no alternate soil extraction procedures that can achieve all of these goals.…”

“…To enable global multi-omic analyses of a single soil sample, an organic solvent extraction protocol based upon chloroform, methanol, and water separations was utilized 10 . This method was originally developed for total lipid extractions 9,11 and more recently was amended for the simultaneous extraction of metabolites, proteins, and lipids from a single sample 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30, enabling less sample quantity and experimental variability 10 .…”

“…Each soil type can be different, however, and in highly organic soil such as peat, the soil debris stays in the middle layer and does not fall to the bottom of the sampling tube. MPLEx provides several advantages when isolating multiple molecule types from the same sample such as 1) smaller sample quantities can be used for multi-omic analyses, 2) multi-omic extractions from the same sample decrease overall experimental variability, and 3) greater numbers of samples can be prepared much faster for higher throughput studies 10 . Together these benefits are vital for providing better measurement capabilities for evaluating soil samples and their complex microbial communities.…”