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Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics Based on Acid Extraction
Open accessPublished version of a preprint
Abstract
SUMMARY Mass spectrometry is the method of choice for deep and comprehensive analysis of proteomes and has become a key technology to support the progress in life science and biomedicine. However, sample preparation in proteomics is not standardized and contributes to a lack of reproducibility. The main challenge is to extract all proteins in a manner that enables efficient digestion into peptides and is compatible with subsequent mass spectrometric analysis. Current methods are based on the idea of removing detergents or chaotropic agents during sample processing, which are essential for protein extraction but interfere with digestion and LC-MS. These multi-step preparations are prone to losses, biases and contaminations, while being time-consuming and labor-intensive. We report a universal detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED) , which is based on a simple three-step procedure, acidification, neutralization and digestion. SPEED is a one-pot method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction. SPEED-based sample processing is highly reproducible, provides exceptional peptide yields and enables preparation even of tissue samples with less than 15 min hands-on time and without any special equipment. Evaluation of SPEED performance revealed, that the number of quantified proteins and the quantitative reproducibility are superior compared to the well-established sample processing protocols FASP, ISD-Urea and SP3 for various sample types, including human cells, bacteria and tissue, even at low protein starting amounts.
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Metadata
- Authors
- Doellinger, J., Schneider, A., Hoeller, M., & Lasch, P.
- Affiliations
- Robert Koch Institute (DE)
- Corresponding author
- Doellinger, Joerg
- Venue
- Molecular & Cellular Proteomics
- Publisher
- Elsevier BV
- Published
- 2020-01
- Volume
- 19
- Issue
- 1
- Pages
- 209-222
- ISSN
- 1535-9476 (ISSN-L)
- PMC
- PMC6944244
- OpenAlex
- W2989663787
- Semantic Scholar
- b2421ccb33741a7fba2821d3b8942779a2eb28be
- Cited by
- 246
- References
- 46
- Citations by year
- 2026: 47 · 2025: 56 · 2024: 49 · 2023: 37 · 2022: 24 · 2021: 21 · 2020: 11
- FWCI
- 5.66
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular & Cellular Proteomics (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Publication server of the Robert Koch Institute (Robert Kock Institute) (submittedVersion, cc-by)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
- Keywords
- acid extraction, protein digestion, trifluoroacetic acid, peptide production, shotgun proteomics, quantitative proteomics, sample preparation reproducibility, proteome coverage
- MeSH terms
- Animals, Bacillus cereus, Chromatography, Liquid, Detergents, HeLa Cells, Humans, Lung, Mice, Inbred C57BL, Peptides, Proteins, Staphylococcus aureus, Trifluoroacetic Acid, Urea, Reproducibility of Results, Proteome, Proteomics, Escherichia coli K12, Mice, Tandem Mass Spectrometry, Proteolysis
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