Impact of Sample Preparation Strategies on the Quantitative Accuracy of Low-Abundance Serum Proteins in Shotgun Proteomics

Abstract

Serum proteomics plays a crucial role in biomarker discovery and disease research, yet the selection of an optimal sample preparation method remains challenging. Evaluating the accuracy of protein quantitation is of major importance and a vital part of a benchmarking study in proteomics, since clinical studies are based on the differential expression of certain proteins that can be used as biomarkers or be indicative of a pathological state. In this study, we performed a direct comparison of 6 widely used serum proteomic sample preparation workflows: In-gel digestion (IGD), Single-Pot Solid-Phase-enhanced Sample Preparation (SP3), Top 14 Abundant Protein Depletion (Top 14), Isopropanol/Trichloroacetic Acid (IPA/TCA) precipitation, PreOmics ENRICH-iST (PreOmics), and Seer Proteograph XT (Seer). Seer and PreOmics demonstrated superior quantitative accuracy, especially for proteins with low abundance in serum, while the Seer enrichment approach provided the highest number of protein identifications (>2000) as measured by an Orbitrap Exploris 480. All methods had median CVs close to or below 20%. This comparative analysis provides a comprehensive resource for selecting the most appropriate serum sample preparation strategy based on specific experimental needs, facilitating human serum proteomic profiling for biomedical research.