• Easy Proteomics Sample Prepara...

Treffer: Easy Proteomics Sample Preparation: Technical Repeatability and Workflow Optimization Across 8 Biological Matrices in a New Core Facility Setting.

Title:
Easy Proteomics Sample Preparation: Technical Repeatability and Workflow Optimization Across 8 Biological Matrices in a New Core Facility Setting.
Authors:
Karousi P; Section of Biochemistry and Molecular Biology, Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens, Greece.; Proteomics Core Facility, School of Science, National and Kapodistrian University of Athens, Athens, Greece., Voumvouraki M; Proteomics Core Facility, School of Science, National and Kapodistrian University of Athens, Athens, Greece.; Laboratory of Analytical Chemistry, Department of Chemistry, School of Science, National & Kapodistrian University of Athens, Athens, Greece., Nikolaou PE; Laboratory of Pharmacology, Department of Pharmacy, School of Health Sciences, National & Kapodistrian University of Athens, Athens, Greece.; Section of Clinical Therapeutics, Department of Medicine, School of Health Sciences, National and Kapodistrian University of Athens, Athens, Greece., Kollias I; Section of Clinical Therapeutics, Department of Medicine, School of Health Sciences, National and Kapodistrian University of Athens, Athens, Greece., Paradeisi F; Proteomics Laboratory, Biomedical Research Foundation, Academy of Athens, Athens, Greece., Sampanai E; Section of Biochemistry and Molecular Biology, Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens, Greece., Gkalea V; Hematology Department, Alexandra General Hospital, Athens, Greece., Morianos I; Host Defense & Fungal Pathogenesis Lab, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Athens, Greece., Zoidakis J; Section of Biochemistry and Molecular Biology, Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens, Greece.; Proteomics Laboratory, Biomedical Research Foundation, Academy of Athens, Athens, Greece., Kastritis E; Section of Clinical Therapeutics, Department of Medicine, School of Health Sciences, National and Kapodistrian University of Athens, Athens, Greece., Thomaidis N; Laboratory of Analytical Chemistry, Department of Chemistry, School of Science, National & Kapodistrian University of Athens, Athens, Greece., Médard G; Proteomics Core Facility, School of Science, National and Kapodistrian University of Athens, Athens, Greece., Courraud J; Proteomics Core Facility, School of Science, National and Kapodistrian University of Athens, Athens, Greece.; Section of Clinical Therapeutics, Department of Medicine, School of Health Sciences, National and Kapodistrian University of Athens, Athens, Greece.
Source:
Proteomics [Proteomics] 2025 Oct; Vol. 25 (20), pp. 15-24. Date of Electronic Publication: 2025 Oct 24.
Publication Type:
Journal Article
Language:
English
Journal Info:
Publisher: Wiley-VCH Country of Publication: Germany NLM ID: 101092707 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1615-9861 (Electronic) Linking ISSN: 16159853 NLM ISO Abbreviation: Proteomics Subsets: MEDLINE
Imprint Name(s):
Original Publication: Weinheim, Germany : Wiley-VCH,
MeSH Terms:
Proteomics*/methods , Proteome*/analysis , Specimen Handling*/methods, Workflow ; Tandem Mass Spectrometry/methods ; Humans ; Chromatography, Liquid/methods ; Reproducibility of Results ; Animals
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Grant Information:
101097094 Horizon Europe ELMUMY Project; 101079217 Horizon Europe STEPUPIORS Project; TAEDR-0541976 General Secretariat for Research and Innovation of Greece, European Union-Next Generation EU, Grant PRO-sCAP; Hellenic Foundation for Research and Innovation (H.F.R.I.), Project ENLIGHTEnMENT; International Myeloma Society Translational Research Award
Contributed Indexing:
Keywords: SPEED protocol; bottom‐up proteomics; diaPASEF; technical repeatability; tryptic peptides
Substance Nomenclature:
0 (Proteome)
Entry Date(s):
Date Created: 20251024 Date Completed: 20251031 Latest Revision: 20251102
Update Code:
20251102
PubMed Central ID:
PMC12576925
DOI:
10.1002/pmic.70064
PMID:
41132037
Database:
MEDLINE

Weitere Informationen

Bottom-up proteomics relies on efficient and repeatable sample preparation for accurate protein identification and precise quantification. This study evaluates the performance of adapted SPEED (Sample Preparation by Easy Extraction and Digestion) protocol, a simplified, detergent-free approach tailored for various biological matrices, including lysis-resistant samples. Protein extraction and denaturation steps were refined for 8 biological matrices enabling standardized, cheap, and scalable proteomics analysis on 96-well plates. For tissue samples requiring downstream applications like Western blotting, we used a low-detergent RIPA buffer. Notably, the protocols demonstrate remarkable down-scalability, enabling robust proteomics measurements from as few as 3000 cells per sample for preparation and even down to 300 cells per LC-MS/MS analysis. Key advancements include a 30-min nanoLC-MS/MS run, achieving a 15-20 samples-per-day throughput, and leveraging the power of diaPASEF using thoroughly optimized DIA-windows to enhance proteome coverage. These adaptations streamline workflows, enabling proteomics analyses in matrices with challenging physical and biochemical properties. This study underscores the importance of early-stage optimization and feasibility testing in proteomics pipelines to inform study design and sample selection. By showcasing robust, scalable adaptations of the SPEED protocol, we provide a foundation for reproducible, high-throughput proteomic studies across diverse biological contexts.
(© 2025 The Author(s). PROTEOMICS published by Wiley‐VCH GmbH.)