Multiple reaction monitoring profiling (MRM profiling): Small molecule exploratory analysis guided by chemical functionality

Small molecules, including metabolites and lipids, provide information on metabolic pathways and active biological processes in living organisms. They are often diagnostic of disease. Current exploratory methods for metabolomics and lipidomics mostly rely on separation using liquid or gas chromatography (LC or GC) coupled with mass spectrometers capable of acquiring high resolution data to generate an enormous data, but at the cost of lengthy processing and data acquisition. Even though many molecules can be identified and quantified by these methods, the laborious protocols for purification, identification, and validation limit the accessible sample chemical information. To improve the speed and efficiency of exploratory metabolomics and lipidomics, multiple reaction monitoring profiling (MRM profiling) has been developed. This strategy involves a three-stage workflow which starts by considering the metabolome as a collection of functional groups. The Discovery Stage interrogates a representative sample mixture for functional groups using the functional group specific precursor ion (Prec) scans and neutral loss (NL) scans. This experiment usually uses a triple quadrupole mass spectrometer without chromatography, i.e. by direct sample infusion. In the second Screening Stage, the main features seen in the Prec and NL scans are organized into lists of precursor ion/product ion transitions (MRMs) which are then used for the fast, specific, and sensitive interrogation of each individual sample. Data analysis by univariate and multivariate statistical methods is used to identify the most informative MRMs and so classify the individual samples. The compounds (biomarkers) which are responsible for the most informative MRMs in particular sample classes can be investigated in an optional third Identification Stage i.e. in a structural identification study. MRM profiling benefits from the much smaller number of functional groups compared to the number of individual metabolites existing in biological samples (where most metabolites are still unknown), resulting in acquisition of a much smaller data set and a shorter analysis time. The application of MRM Profiling to several biological and clinical problems is used to illustrate its features.