MASS SPECTROMETRY-BASED MULTI-OMICS STRATEGIES TO SUPPORT THE DEVELOPMENT OF NEW THERAPEUTIC APPROACHES

THE RAPID EVOLUTION OF OMICS SCIENCES IS PROFOUNDLY TRANSFORMING BIOMEDICAL RESEARCH AND ACCELERATING THE SHIFT TOWARD PRECISION AND PERSONALIZED MEDICINE. AMONG OMICS DISCIPLINES, METABOLOMICS HOLDS A DISTINCTIVE ROLE AS IT PROVIDES A DIRECT FUNCTIONAL SNAPSHOT OF BIOCHEMICAL ACTIVITY, INTEGRATING GENETIC, EPIGENETIC, AND ENVIRONMENTAL INFLUENCES. LIQUID CHROMATOGRAPHY–HIGH RESOLUTION MASS SPECTROMETRY (LC-HRMS) HAS BECOME THE GOLD STANDARD FOR METABOLOMICS AND LIPIDOMICS; HOWEVER, ACHIEVING COMPREHENSIVE METABOLOME COVERAGE REMAINS CHALLENGING DUE TO THE VAST CHEMICAL DIVERSITY AND DYNAMIC CONCENTRATION RANGE OF SMALL MOLECULES. THIS PHD PROJECT FOCUSES ON THE DEVELOPMENT AND OPTIMIZATION OF ADVANCED LC-MS-BASED MULTI OMICS STRATEGIES DESIGNED TO IMPROVE ANALYTICAL PERFORMANCE, EXPAND METABOLOME COVERAGE, AND ENABLE HIGH SENSITIVITY PROFILING OF LIMITED BIOLOGICAL MATERIAL. IN THE FIRST PHASE, REVERSED PHASE (RP) AND HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY (HILIC) WORKFLOWS WERE SYSTEMATICALLY OPTIMIZED FOR UNTARGETED METABOLOMICS USING HUMAN PLASMA AS A COMPLEX REFERENCE MATRIX. A PEAK QUALITY–BASED EVALUATION FRAMEWORK WAS IMPLEMENTED TO ASSESS CHROMATOGRAPHIC ROBUSTNESS, REPRODUCIBILITY, AND OVERALL DATA QUALITY. THE COMBINED USE OF COMPLEMENTARY RP AND HILIC PLATFORMS MARKEDLY ENHANCED METABOLOME COVERAGE WHILE MAINTAINING RIGOROUS ANALYTICAL STANDARDS. THE OPTIMIZED PLATFORM WAS NEXT APPLIED TO A CLINICALLY RELEVANT STUDY OF HEPATOCELLULAR CARCINOMA (HCC) IN PATIENTS WITH HEPATITIS C VIRUS (HCV) INFECTION. INTEGRATED UNTARGETED METABOLOMICS AND LIPIDOMICS ANALYSES REVEALED METABOLIC SIGNATURES LINKED TO MITOCHONDRIAL DYSFUNCTION, ALTERED FATTY ACID OXIDATION, AND MEMBRANE LIPID REMODELING. MULTIVARIATE MODELING DEMONSTRATED SUPERIOR DIAGNOSTIC PERFORMANCE COMPARED WITH ALPHA FETOPROTEIN (AFP), INCLUDING IN AFP NEGATIVE CASES. TO FACILITATE CLINICAL TRANSLATION, A RAPID TARGETED HILIC–HRMS ASSAY WAS DEVELOPED ON A QUADRUPOLE–ORBITRAP SYSTEM OPERATING IN MULTIPLEXED SINGLE ION MONITORING MODE. FOCUSING ON ACYLCARNITINES AND LYSOPHOSPHATIDYLCHOLINES, THE METHOD ACHIEVED HIGH SENSITIVITY, QUANTITATIVE ACCURACY, AND ROBUSTNESS, CONFIRMING THE DIAGNOSTIC RELEVANCE OF THE IDENTIFIED BIOMARKERS. RECOGNIZING THE GROWING NEED FOR HIGH SENSITIVITY ANALYSES FROM LOW INPUT SAMPLES, CHROMATOGRAPHIC MINIATURIZATION WAS FURTHER EXPLORED. A SYSTEMATIC COMPARISON OF CONVENTIONAL 2.1 MM I.D. AND MICROBORE 1.0 MM I.D. UHPLC CONFIGURATIONS REVEALED SUBSTANTIAL IMPROVEMENTS IN SENSITIVITY, DETECTION LIMITS, REPEATABILITY, AND METABOLOME COVERAGE WITHOUT COMPROMISING ROBUSTNESS OR THROUGHPUT. FINALLY, THE PRINCIPLES OF ANALYTICAL MINIATURIZATION WERE EXTENDED TO MICRODISSECTED THYROID TUMOR TISSUES THROUGH A DEEP VISUAL PROTEOMICS WORKFLOW, ENABLING DEEP MOLECULAR PROFILING FROM EXTREMELY LIMITED SAMPLE MATERIAL. COLLECTIVELY, THIS WORK ESTABLISHES AN INTEGRATED AND SENSITIVITY DRIVEN MULTI OMICS FRAMEWORK THAT COMBINES CHROMATOGRAPHIC OPTIMIZATION, HIGH RESOLUTION MASS SPECTROMETRY, TARGETED ASSAY DEVELOPMENT, AND WORKFLOW MINIATURIZATION. THESE ADVANCEMENTS SIGNIFICANTLY EXPAND METABOLOME COVERAGE, IMPROVE ANALYTICAL ROBUSTNESS, AND SUPPORT BIOMARKER DISCOVERY AND TRANSLATIONAL RESEARCH IN PRECISION MEDICINE.