Isotope‐Labeled Chemoselective Probes for Labeling, Separation, and Comprehensive Quantitative Analysis of Sub‐Metabolome

Abstract The significance of small molecule metabolites as biomarkers for disease diagnosis and prognosis is growing increasingly evident, necessitating the development of highly sensitive qualitative and quantitative methods. Herein, multi‐chemoselective probes are synthesized and applied for profiling metabolites, including carboxyl, phosphate, hydroxyl, amino, thiol, and carbonyl compounds. This approach seamlessly integrates magnetic solid‐phase materials, orthogonal cleavage sites, isotopic tags, and selective coupling sites, minimizes matrix interference, and enhances quantitative accuracy. Meanwhile, a homemade program, High‐Resolution Isotope‐Assisted Identification and Quantitative (HRIAI Quant ) is developed to process the data, which adeptly filters through 33,874 ion pairs present in human serum, leading to the identification of 701 known metabolites and a remarkable 1,062 potential novel ones. This method is successfully applied to analyze metabolites in multiple brain regions of SAMP8 and SAMR1 models, offering a novel tool for Alzheimer's disease research.