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

Yan Chen; Jennifer W Gin; Ying Wang; Markus de Raad; Stephen Tan; Nathan J Hillson; Trent R Northen; Paul D Adams; Christopher J Petzold

doi:10.1371/journal.pone.0288102

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

PLoS One. 2023 Jul 7;18(7):e0288102. doi: 10.1371/journal.pone.0288102. eCollection 2023.

Authors

Yan Chen^{1

2

3}, Jennifer W Gin^{1

2

3}, Ying Wang⁴, Markus de Raad⁴, Stephen Tan^{1

2

3}, Nathan J Hillson^{1

2

3}, Trent R Northen^{2

4}, Paul D Adams^{2

5

6}, Christopher J Petzold^{1

2

3}

Affiliations

¹ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
² DOE Joint BioEnergy Institute, Emeryville, California, United States of America.
³ DOE Agile BioFoundry, Emeryville, California, United States of America.
⁴ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
⁵ Department of Bioengineering, University of California Berkeley, Berkeley, California, United States of America.
⁶ Molecular Biophysics and Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.

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.

Copyright: © 2023 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

MeSH terms

Acetone* / chemistry
Indicators and Reagents
Proteins
Proteomics* / methods

Substances

Acetone
Proteins
Indicators and Reagents

Grants and funding

The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The views and opinions of the authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. The proof-of-concept work with mono-cultures and resources were part of the Joint BioEnergy Institute (JBEI; http://www.jbei.org), the extension of the procedure and identification of the sources of error were part of the Agile BioFoundry (ABF; http://agilebiofoundry.org), and the application to synthetic microbial communities was part of Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA; http://enigma.lbl.gov), a Science Focus Area led by Lawrence Berkeley National Laboratory. This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research (JBEI and ENIGMA) and through the Bioenergy Technology Office within the DOE Office of Energy Efficiency and Renewable Energy (ABF) through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy.