title: Integrated microfluidic chip and online SCX separation allows untargeted nanoscale metabolomic and peptidomic profiling.
abstract: 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.
authors.affiliation: Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.
authors.forename: Ravi
authors.initials: R
authors.lastname: Tharakan
authors.forename: Dingyin
authors.initials: D
authors.lastname: Tao
authors.forename: Ceereena
authors.initials: C
authors.lastname: Ubaida-Mohien
authors.forename: Rhoel R
authors.initials: RR
authors.lastname: Dinglasan
authors.forename: David R
authors.initials: DR
authors.lastname: Graham
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D056656:Lab-On-A-Chip Devices; D055432:Metabolomics; D010455:Peptides / Q000737:chemistry*
chemical_list: D010455:Peptides
keywords: integrative biology; liquid chromatography; mass spectrometry; metabolomics; strong cation exchange; systems biology