Cross-Platform Evaluation Reveals Sample Preparation-Driven Biological Biases despite Advances in Mass Spectrometry Performance in Serum Proteomics

Abstract Advances in mass spectrometry (MS) instrumentation and protein extraction technologies are reshaping proteomics; however, whether improved analytical performance alters the biological information accessible from different sample preparation strategies remains unclear. Here, we performed a cross-platform evaluation using identical benchmark serum samples previously analyzed on an Orbitrap Exploris 480 platform, including the newly introduced Seer Proteograph ONE workflow, to determine how MS advances and sample preparation influence analytical performance and biological interpretation. Across all preparation strategies, the timsTOF HT improved analytical performance by increasing protein and peptide identifications, reducing missing values, and improving linearity in spike-in experiments, particularly for low-abundance proteins. Despite these improvements, sample preparation remained the major determinant of the accessible serum proteome. Functional enrichment analysis revealed reproducible workflow-specific biases across MS platforms: nanoparticle-based enrichment expanded access to a broader range of serum proteins and improved detection of clinically relevant signaling-associated proteins, including cytokine- and chemokine-related marker classes, whereas the depletion-based strategy showed a distinct functional profile enriched for receptor-related activities. Together, these findings demonstrate that MS advances enhance analytical performance but do not eliminate sample preparation-driven differences in biological accessibility. This study provides a framework for selecting serum proteomics workflows according to specific biomarker and pathway objectives.