biostudies-literatureKatsavelis DDutch Research Council (NWO)European Commission4792-4803https://www.ebi.ac.uk/biostudies/studies/S-EPMC12418494biostudies-literatureUnknown24(9)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 (SeJournal of proteome researchImpact of Sample Preparation Strategies on the Quantitative Accuracy of Low-Abundance Serum Proteins in Shotgun Proteomics.PMC12418494184.034.019JTFN-00102Permentier HPHorvatovich PKatsavelis Dvan der Hart MGCWolters JCCremers TIFHfalseImpact of Sample Preparation Strategies on the Quantitative Accuracy of Low-Abundance Serum Proteins in Shotgun Proteomics.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 (Se2025-01-01T00:00:00Z2025 Sep2026-05-26T20:15:52.486Z2026-05-24T03:07:44.201ZS-EPMC124184944085758510.1021/acs.jproteome.5c00572