A Standardized Metabolomics Protocol For Analyzing Exercise-Induced Metabolic Shifts In Elite Boxers By Liquid Chromatography-Mass Spectrometry

Summary

This protocol describes a standardized serum metabolomics workflow based on liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) and multivariate data analysis for profiling acute and short-term recovery-related metabolic shifts in elite male boxers.

Abstract

Elite boxing induces rapid metabolic changes that are not fully captured by conventional physiological measurements. A standardized untargeted serum metabolomics workflow based on liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) was applied to samples collected before sparring, immediately after sparring, and 24 h after sparring in seven elite male boxers. The workflow included standardized sample collection, pooled quality-control monitoring, metabolite profiling, multivariate statistical analysis, and pathway interpretation. Acute sparring was associated with changes in metabolites related to glycolysis and gluconeogenesis, whereas the 24-h timepoint was associated with sulfur metabolism.

Phosphatidylinositol PI(16:0/18:2(9Z,12Z)) showed strong discrimination of the immediate post-sparring state, and thiosulfate was associated with the 24-h recovery state. These findings support the use of this workflow for reproducible profiling of exercise-related metabolic changes in this cohort of seven elite male boxers. The protocol is intended for controlled small-cohort studies of acute exercise and short-term recovery and emphasizes reproducible sample handling, pooled quality-control monitoring, and interpretable downstream analysis.

Introduction

Elite boxing, characterized by three rounds of 2 min of high-intensity sparring, relies on a combination of anaerobic and aerobic metabolism and triggers rapid shifts in energy substrates, oxidative stress mediators, and tissue repair-related metabolites. Traditional assessments (blood lactate and heart rate) only capture isolated physiological endpoints, failing to resolve the systemic, interconnected metabolic changes that span “pre-sparring baseline → post-sparring acute stress → 24-h recovery.”

Untargeted metabolomics, enabled by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry, addresses ....

Protocol

Perform all procedures in accordance with approval from the Institutional Ethics Committee for Human Research of Capital University of Physical Education and Sports (approval no. 2025A005).

CAUTION: Treat all human blood specimens as potentially infectious. Wear gloves, a laboratory coat, and eye protection. Perform venous blood collection, serum handling, and waste disposal in accordance with institutional biosafety procedures. Decontaminate work surfaces after sample handling.

CAUTION: Methanol, acetonitrile, and formic acid are hazardous chemicals. Handle solvent preparation, extraction, and reconstitution ste....

Results

Quality Control (QC) Validation of LC-QTOF-MS/MS Data

To evaluate signal stability and reproducibility, repeated injections of the pooled QC sample were analyzed in both ion modes. Spearman correlation coefficients among pooled-QC injections exceeded 0.8 in both ion modes, which indicated stable instrument performance across the analytical sequence. PCA of all study samples and QC injections showed tight clustering of QC injections and no clear outliers, which supported accept.......

Discussion

This standardized workflow characterized metabolite changes associated with elite boxing sparring and short-term recovery in a small, controlled cohort. The pooled-QC injections, QC-based correlation analysis, PCA clustering, and multivariate analyses together supported acceptable analytical stability. The protocol's success depended primarily on four factors: maintaining sparring intensity within 80%–85% of maximum heart rate, processing blood samples within 1 h after collection, limiting each sample to no mor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents	Acetonitrile	Merck	LC-MS grade, CAS 75-05-8
Reagents	Methanol	Merck	LC-MS grade, CAS 67-56-1
Reagents	Formic acid	TCI	LC-MS grade, CAS 64-18-6
Consumables	VACUETTE TUBE CAT Serum Clot Activator	Greiner Bio-One	8 mL serum clot activator tube
Consumables	Nalgene Cryogenic Vial	Thermo Fisher Scientific	1.5 mL, external thread
Consumables	Protein LoBind Tube	Eppendorf	Cat. No. 0030108442, 1.5 mL
Glassware	Screw-top glass vial with insert	Agilent	2 mL vial with 200 μL glass insert; insert Cat. No. 5183-2090
Consumables	Millex-GP Filter	Merck	Cat. No. SLGP033RS, 0.22 μm membrane
Instruments	Custom Boxing Ring 6x6 m	Muay Thai Sport	6 × 6 m training ring
Consumables	Pro Style 2 Boxing Gloves	Everlast	16 oz
Instruments	Polar H10	Polar	Chest-strap heart-rate sensor
Instruments	quasar	h/p/cosmos	Motorized treadmill
Instruments	Centrifuge 5425 R	Eppendorf	Refrigerated microcentrifuge
Instruments	Bransonic CPX2800	Emerson / Branson	40 kHz digital ultrasonic bath
Instruments	Savant SpeedVac SPD1030	Thermo Fisher Scientific	Integrated vacuum concentrator
Instruments	VWR Mini Vortex Mixer	Avantor / VWR	Cat. No. 10153-688
Instruments	TSX ULT Freezer	Thermo Fisher Scientific	Model TSX50086A
Instruments	ACQUITY UPLC I-Class PLUS	Waters	UHPLC system
Instruments	Xevo G2-XS QTof	Waters	QTOF mass spectrometer
Instruments	ACQUITY UPLC HSS T3 Column	Waters	P/N 176001132, 1.8 μm, 2.1 × 100 mm
Software	MassLynx	Waters	Version 4.2
Software	Progenesis QI	Waters	Version 2.3
Software	R	R Foundation for Statistical Computing	Version 3.6.1
Software	prcomp	base R	Base R function
Software	ropls	Bioconductor	R package
Software	pheatmap	CRAN	R package
Software	corrplot	CRAN	R package
Software	kmeans	base R	Base R function
Software	NbClust	CRAN	R package
Software	venn	CRAN	R package
Software	MetaboAnalystR	CRAN / Xia Lab	Version 1.0
Software	pROC	CRAN	Version 1.15.0
Software	clusterProfiler	Bioconductor	Bioconductor package
Databases	Human Metabolome Database (HMDB)	HMDB	Online database
Databases	Kyoto Encyclopedia of Genes and Genomes (KEGG)	KEGG	Online database
Databases	LIPID MAPS	LIPID MAPS	Online database