Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
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, wemore »
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO)
- OSTI Identifier:
- 1988658
- Alternate Identifier(s):
- OSTI ID: 2228891
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Published Article
- Journal Name:
- PLoS ONE
- Additional Journal Information:
- Journal Name: PLoS ONE Journal Volume: 18 Journal Issue: 7; Journal ID: ISSN 1932-6203
- Publisher:
- Public Library of Science (PLoS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Chen, Yan, Gin, Jennifer W., Wang, Ying, de Raad, Markus, Tan, Stephen, Hillson, Nathan J., Northen, Trent R., Adams, Paul D., Petzold, Christopher J., and Nasim, ed., Faiz ul-Hassan. Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format. United States: N. p., 2023.
Web. doi:10.1371/journal.pone.0288102.
Chen, Yan, Gin, Jennifer W., Wang, Ying, de Raad, Markus, Tan, Stephen, Hillson, Nathan J., Northen, Trent R., Adams, Paul D., Petzold, Christopher J., & Nasim, ed., Faiz ul-Hassan. Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format. United States. https://doi.org/10.1371/journal.pone.0288102
Chen, Yan, Gin, Jennifer W., Wang, Ying, de Raad, Markus, Tan, Stephen, Hillson, Nathan J., Northen, Trent R., Adams, Paul D., Petzold, Christopher J., and Nasim, ed., Faiz ul-Hassan. Fri .
"Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format". United States. https://doi.org/10.1371/journal.pone.0288102.
@article{osti_1988658,
title = {Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format},
author = {Chen, Yan and Gin, Jennifer W. and Wang, Ying and de Raad, Markus and Tan, Stephen and Hillson, Nathan J. and Northen, Trent R. and Adams, Paul D. and Petzold, Christopher J. and Nasim, ed., Faiz ul-Hassan},
abstractNote = {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.},
doi = {10.1371/journal.pone.0288102},
journal = {PLoS ONE},
number = 7,
volume = 18,
place = {United States},
year = {Fri Jul 07 00:00:00 EDT 2023},
month = {Fri Jul 07 00:00:00 EDT 2023}
}
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https://doi.org/10.1371/journal.pone.0288102
https://doi.org/10.1371/journal.pone.0288102
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