Quantification of serum myoglobin by liquid chromatography-isotope dilution mass spectrometry

Human myoglobin (hMYO) is a sensitive early biomarker for acute myocardial infarction, rising within 1~3 h after myocardial injury and offering high predictive value for excluding infarction. To standardize its measurement, a precise quantitative method combining magnetic bead extraction with liquid chromatography-isotope dilution tandem mass spectrometry (LC‑IDMS/MS) was established. ¹⁵NLabeled myoglobin was spiked into serum as an internal standard, and magnetic beads coated with monoclonal antibodies (mAbs) against hMYO were used for the extraction of hMYO from serum. The mAb employed in this study demonstrated comparable binding affinity for both native myoglobin and its isotopically labeled counterpart, as confirmed by surface plasmon resonance measurements. After extraction, the magnetic beads were washed and digested, and two signature peptides, VEADIPGHGQEVLIR (VR) and HGATVLTALGGILK (HGK), were selected for quantification of hMYO. The incubation time, bead and enzyme amounts, and digestion time were optimized to establish optimal sample treatment conditions. Digested peptides were analyzed by LC‑IDMS/MS and recovery based on the VR peptide was 95.4-101.6% (RSD = 2.3%), and based on the HGK peptide was 100.1-105.5% (RSD = 2.0%). Limits of detection and quantification of hMYO were 1.04 ng/g and 3.43 ng/g by VR, and 1.45 ng/g and 4.74 ng/g by HGK. The proposed method enables accurate serum hMYO quantification, supports reference material development and clinical standardization, and also provides an example for MS‑based quantification of clinical protein biomarkers.