Label-free quantitative proteomics analysis of sea cucumber (Apostichopus japonicus) from different origins using data-independent acquisition mass spectrometry

Sea cucumbers (<i>Apostichopus japonicus</i>) from Liaoning, China command premium market value due to superior collagen integrity, yet the proteomic basis of this geographical differentiation remains unclear. Current geographic proteomic studies using data-dependent acquisition (DDA) mass spectrometry face stochastic ion selection bias, poor low-abundance reproducibility and limited coverage. Here, we established a custom label-free data-independent acquisition (DIA) workflow to characterize proteomic profiles of 200 sea cucumbers (80 Liaoning, 120 non-Liaoning). Our approach identified 6278 proteins and discovered 64 differentially expressed proteins (DEPs), including arginase (A0A141R8A8) and glutamine synthetase (A0A076VCY3) as potential authentication markers. Functional annotation highlighted arginine biosynthesis (<i>P</i> = 0.00034) as the pivotal pathway, alongside two-component system and necroptosis, indicating nitrogen homeostasis prioritization and stress adaptation in Liaoning specimens. This study provides a novel DIA-based framework for discovering the proteomic basis of geographical differentiation, linking nitrogen metabolism to regional quality differences in marine foods and identifying potential molecular targets for future authentication efforts.