ArticlePloS one2023
Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format.
Article in PloS one, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 23 papers.
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23 citing papers in PubMed, 33 citations in OpenAlex.
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- Proteomic Analysis ofInternational journal of molecular sciences · 2025Article
- Automation and machine learning drive rapid optimization of isoprenol production in Pseudomonas putida.Nature communications · 2025Article
- Engineering Polyketide Stereocenters with Ketoreductase Domain Exchanges.Journal of the American Chemical Society · 2025Article
- Phosphoproteomic Analysis Reveals Impairment of Rice Germination by Chloramphenicol.Plants (Basel, Switzerland) · 2025Article
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- Alternate routes to acetate tolerance lead to varied isoprenol production from mixed carbon sources inApplied and environmental microbiology · 2025Article
- Addressing genome scale design tradeoffs in Pseudomonas putida for bioconversion of an aromatic carbon source.NPJ systems biology and applications · 2025Article
- Emergence and disruption of cooperativity in a denitrifying microbial community.The ISME journal · 2025Article
- Verazine biosynthesis from simple sugars in engineered Saccharomyces cerevisiae.Metabolic engineering · 2024Article
- Expression of dehydroshikimate dehydratase in poplar induces transcriptional and metabolic changes in the phenylpropanoid pathway.Journal of experimental botany · 2024Article
- Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit.Nature communications · 2024Article
- Top-down and bottom-up microbiome engineering approaches to enable biomanufacturing from waste biomass.Journal of industrial microbiology & biotechnology · 2024Review
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Authors and funding
9 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes (e.g., Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup. The yield of protein using this protocol increases linearly with respect to the amount of starting biomass from 0.5-2.0 OD*mL of cells. By using a bench-top automated liquid dispenser, a cost-effective and environmentally-friendly option to eliminating pipette tips and reducing reagent waste, the protocol takes approximately 30 minutes to extract protein from 96 samples. Tests on mock mixtures showed expected results that the biomass composition structure is in close agreement with the experimental design. Lastly, we applied the protocol for the composition analysis of a synthetic community of environmental isolates grown on two different media. This protocol has been developed to facilitate rapid, low-variance sample preparation of hundreds of samples and allow flexibility for future protocol development.
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