Integrated Microfluidic Chip and Online SCX Separation Allows Untargeted Nanoscale Metabolomic and Peptidomic Profiling

Metabolomics and peptidomics are systems biology approaches in which broad populations of molecular species produced in a cell or tissue sample are identified and quantified. These two molecular populations, metabolites and peptides, can be extracted from tissues in a similar fashion, and we therefore have here developed an integrated platform for their extraction and characterization. This was accomplished by liquid–liquid extraction of peptides and metabolites from tissue samples and online strong cation exchange (SCX) separation to allow characterization of each population individually. The platform was validated both by a mixed set of purified standards and by an analysis of splenic tissue from SIV-infected macaques, showing both good reproducibility in chromatography, with relative standard deviation (RSD) of hold time less than 0.4%, and clear separation of charge state, with ∼95% of molecular features in SCX separated runs at charge states of +1 or +2. Finally, we used this platform to analyze the physiological response to infection in the spleen, showing that the spleen contains an abundance of hemoglobin-derived peptides, which do not appear to change in response to infection, and that there appears to be a large and variable metabolic response to infection. We therefore present a method for peptidomic and metabolomic profiling which is simple, robust, and easy to implement.