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Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format

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 gro...

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Autores principales: Chen, Yan, Gin, Jennifer W., Wang, Ying, de Raad, Markus, Tan, Stephen, Hillson, Nathan J., Northen, Trent R., Adams, Paul D., Petzold, Christopher J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10328223/
https://www.ncbi.nlm.nih.gov/pubmed/37418444
http://dx.doi.org/10.1371/journal.pone.0288102
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author Chen, Yan
Gin, Jennifer W.
Wang, Ying
de Raad, Markus
Tan, Stephen
Hillson, Nathan J.
Northen, Trent R.
Adams, Paul D.
Petzold, Christopher J.
author_facet Chen, Yan
Gin, Jennifer W.
Wang, Ying
de Raad, Markus
Tan, Stephen
Hillson, Nathan J.
Northen, Trent R.
Adams, Paul D.
Petzold, Christopher J.
author_sort Chen, Yan
collection PubMed
description 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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spelling pubmed-103282232023-07-08 Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format Chen, Yan Gin, Jennifer W. Wang, Ying de Raad, Markus Tan, Stephen Hillson, Nathan J. Northen, Trent R. Adams, Paul D. Petzold, Christopher J. PLoS One Lab Protocol 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. Public Library of Science 2023-07-07 /pmc/articles/PMC10328223/ /pubmed/37418444 http://dx.doi.org/10.1371/journal.pone.0288102 Text en © 2023 Chen et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Lab Protocol
Chen, Yan
Gin, Jennifer W.
Wang, Ying
de Raad, Markus
Tan, Stephen
Hillson, Nathan J.
Northen, Trent R.
Adams, Paul D.
Petzold, Christopher J.
Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
title Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
title_full Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
title_fullStr Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
title_full_unstemmed Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
title_short Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
title_sort alkaline-sds cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
topic Lab Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10328223/
https://www.ncbi.nlm.nih.gov/pubmed/37418444
http://dx.doi.org/10.1371/journal.pone.0288102
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