| Autor: |
Mousseau CB; Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA. mchampio@nd.edu., Pierre CA; Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA. mchampio@nd.edu., Hu DD; Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA. mchampio@nd.edu., Champion MM; Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA. mchampio@nd.edu.; Berthiaume Institute for Precision Health, University of Notre Dame, Notre Dame, IN 46556, USA. |
| Jazyk: |
angličtina |
| Zdroj: |
Analytical methods : advancing methods and applications [Anal Methods] 2023 Feb 16; Vol. 15 (7), pp. 916-924. Date of Electronic Publication: 2023 Feb 16. |
| DOI: |
10.1039/d2ay01549h |
| Abstrakt: |
Complete enzymatic digestion of proteins for bottom-up proteomics is substantially improved by use of detergents for denaturation and solubilization. Detergents however, are incompatible with many proteases and highly detrimental to LC-MS/MS. Recently; filter-based methods have seen wide use due to their capacity to remove detergents and harmful reagents prior to digestion and mass spectrometric analysis. We hypothesized that non-specific protein binding to negatively charged silica-based filters would be enhanced by addition of lyotropic salts, similar to DNA purification. We sought to exploit these interactions and investigate if low-cost DNA purification spin-filters, 'Minipreps,' efficiently and reproducibly bind proteins for digestion and LC-MS/MS analysis. We propose a new method, Miniprep Assisted Proteomics (MAP), for sample preparation. We demonstrate binding capacity, performance, recovery and identification rates for proteins and whole-cell lysates using MAP. MAP recovered equivalent or greater protein yields from 0.5-50 μg analyses benchmarked against commercial trapping preparations. Nano UHPLC-MS/MS proteome profiling of lysates of Escherichia coli had 99.3% overlap vs. existing approaches and reproducibility of replicate minipreps was 98.8% at the 1% FDR protein level. Label Free Quantitative proteomics was performed and 91.2% of quantified proteins had a %CV <20% (2044/2241). Miniprep Assisted Proteomics can be performed in minutes, shows low variability, high recovery and proteome depth. This suggests a significant role for adventitious binding in developing new proteomics sample preparation techniques. MAP represents an efficient, ultra-low-cost alternative for sample preparation in a commercially obtainable device that costs ∼$0.50 (USD) per miniprep. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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