Online Nanoflow Two-Dimension Comprehensive Active Modulation Reversed Phase-Reversed Phase Liquid Chromatography High-Resolution Mass Spectrometry for Metaproteomics of Environmental and Microbiome Samples.

Metaproteomics is a powerful analytical approach that can assess the functional capabilities deployed by microbial communities in both environmental and biomedical microbiome settings. Yet, the mass spectra resulting from these mixed biological communities are challenging to obtain due to the high number of low intensity peak features. The use of multiple dimensions of chromatographic separation prior to mass spectrometry analyses has been applied to proteomics previously but can require increased sampling handling and instrument time. Here, we demonstrate an automated online comprehensive active modulation two-dimensional liquid chromatography method for metaproteome sample analysis. A high pH PLRP-S column was used in the first dimension followed by low pH separation in the second dimension using dual modulating C18 traps and a C18 column. This method increased the number of unique peptides found in ocean metaproteome samples by more than 50% when compared to a one-dimension separation while using the same amount of sample and instrument time.