1. Concepts of m/z, RT, and Exact Mass
(1) m/z: Measured mass-to-charge ratio of an analyte, representing the ratio of molecular weight to charge number detected in mass spectrometry.
(2) RT: Retention time, the time at which a compound elutes from a chromatographic column, typically reported in seconds (s) or minutes (min). It reflects the retention behavior of compounds in the chromatographic system.
(3) Exact Mass: The precise molecular weight calculated from the molecular formula, excluding any adducts or modifications.
2. Concepts of TIC, BPC, and MS/MS Spectrum
(1) TIC (Total Ion Chromatogram): A chromatographic profile constructed by plotting the summed intensity of all detected ions at each time point during analysis, representing the overall ion response of the sample.
(2) BPC (Base Peak Chromatogram): A chromatogram generated by continuously plotting the intensity of the most abundant ion (base peak) at each time point throughout separation.
(3) MS/MS Spectrum (Tandem Mass Spectrum): A spectrum produced by further fragmenting a selected precursor ion from full-scan MS, displaying the m/z and relative intensity of product ions.
In brief:
Both TIC and BPC are chromatograms with retention time (RT) as the x-axis and ion intensity as the y-axis.
An MS/MS spectrum displays fragmentation information, with m/z as the x-axis and product ion intensity as the y-axis.
3. Concepts of FC, log₂FC, and VIP
(1) FC (Fold Change): The ratio of metabolite abundance between two biological groups, used to quantify the magnitude of differential expression.
For comparison A vs B, FC = Mean(A) / Mean(B).
log₂FC > 0: upregulation (A is higher than B)
log₂FC < 0: downregulation (A is lower than B)
(2) VIP (Variable Importance in Projection): A weighted index derived from the (O)PLS‑DA model that reflects the contribution of each metabolite to group discrimination. It prioritizes biologically meaningful differential metabolites by quantifying explanatory power for sample classification.
4. How to Identify Biomarkers
(1) Quantitative level: Screen differential metabolites using VIP, FC, and p-value simultaneously. Prioritize metabolites with high VIP, large |FC|, small p-value, and good intra-group reproducibility.
(2) Functional level: Select metabolites from pathways or molecular classes relevant to the research objective that show strong differences and good repeatability.
(3) Literature‑guided selection: Identify metabolites reported to be associated with the phenotype or experimental condition, then verify their expression levels in the dataset.
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