Modular automated bottom-up proteomic sample preparation for high-throughput applications
Abstract
Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated liquid handler systems are increasingly used to overcome these issues for many of the sample preparation steps. Here, we detail a step-by-step protocol to prepare samples for bottom-up proteomic analysis for Gram-negative bacterial and fungal cells. The full modular protocol consists of three optimized protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of protein from 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 Pseudomonas putida and Rhodosporidium toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without themore »
- 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)
- OSTI Identifier:
- 1846504
- Alternate Identifier(s):
- OSTI ID: 1891281
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Published Article
- Journal Name:
- PLoS ONE
- Additional Journal Information:
- Journal Name: PLoS ONE Journal Volume: 17 Journal Issue: 2; Journal ID: ISSN 1932-6203
- Publisher:
- Public Library of Science (PLoS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; automation; sample preparation; proteomics; protein quantification; biomek; Lowry assay; cell lysis; normalization
Citation Formats
Chen, Yan, Kaplan Lease, Nurgul, Gin, Jennifer W., Ogorzalek, Tadeusz L., Adams, Paul D., Hillson, Nathan J., Petzold, Christopher J., and Koomen, ed., John Matthew. Modular automated bottom-up proteomic sample preparation for high-throughput applications. United States: N. p., 2022.
Web. doi:10.1371/journal.pone.0264467.
Chen, Yan, Kaplan Lease, Nurgul, Gin, Jennifer W., Ogorzalek, Tadeusz L., Adams, Paul D., Hillson, Nathan J., Petzold, Christopher J., & Koomen, ed., John Matthew. Modular automated bottom-up proteomic sample preparation for high-throughput applications. United States. https://doi.org/10.1371/journal.pone.0264467
Chen, Yan, Kaplan Lease, Nurgul, Gin, Jennifer W., Ogorzalek, Tadeusz L., Adams, Paul D., Hillson, Nathan J., Petzold, Christopher J., and Koomen, ed., John Matthew. Fri .
"Modular automated bottom-up proteomic sample preparation for high-throughput applications". United States. https://doi.org/10.1371/journal.pone.0264467.
@article{osti_1846504,
title = {Modular automated bottom-up proteomic sample preparation for high-throughput applications},
author = {Chen, Yan and Kaplan Lease, Nurgul and Gin, Jennifer W. and Ogorzalek, Tadeusz L. and Adams, Paul D. and Hillson, Nathan J. and Petzold, Christopher J. and Koomen, ed., John Matthew},
abstractNote = {Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated liquid handler systems are increasingly used to overcome these issues for many of the sample preparation steps. Here, we detail a step-by-step protocol to prepare samples for bottom-up proteomic analysis for Gram-negative bacterial and fungal cells. The full modular protocol consists of three optimized protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of protein from 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 Pseudomonas putida and Rhodosporidium toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with protein relative quantity variance (CV%) below 15%.},
doi = {10.1371/journal.pone.0264467},
journal = {PLoS ONE},
number = 2,
volume = 17,
place = {United States},
year = {Fri Feb 25 00:00:00 EST 2022},
month = {Fri Feb 25 00:00:00 EST 2022}
}
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1371/journal.pone.0264467
https://doi.org/10.1371/journal.pone.0264467
Other availability
Search WorldCat to find libraries that may hold this journal
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Ultra Fast Trypsin Digestion of Proteins by High Intensity Focused Ultrasound
journal, August 2005
- López-Ferrer, D.; Capelo, J. L.; Vázquez, J.
- Journal of Proteome Research, Vol. 4, Issue 5
Highly Reproducible Automated Proteomics Sample Preparation Workflow for Quantitative Mass Spectrometry
journal, November 2017
- Fu, Qin; Kowalski, Michael P.; Mastali, Mitra
- Journal of Proteome Research, Vol. 17, Issue 1
Cost-Effective Automated Preparation of Serum Samples for Reproducible Quantitative Clinical Proteomics
journal, April 2019
- Lee, Jihyeon; Kim, Hyunsoo; Sohn, Areum
- Journal of Proteome Research, Vol. 18, Issue 5
Automated sample preparation with SP 3 for low‐input clinical proteomics
journal, January 2020
- Müller, Torsten; Kalxdorf, Mathias; Longuespée, Rémi
- Molecular Systems Biology, Vol. 16, Issue 1
Automated “Cells-To-Peptides” Sample Preparation Workflow for High-Throughput, Quantitative Proteomic Assays of Microbes
journal, August 2019
- Chen, Yan; Guenther, Joel M.; Gin, Jennifer W.
- Journal of Proteome Research, Vol. 18, Issue 10
A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry
journal, July 2017
- Potriquet, Jeremy; Laohaviroj, Marut; Bethony, Jeffrey M.
- PLOS ONE, Vol. 12, Issue 7
A high-throughput sample preparation method for cellular proteomics using 96-well filter plates
journal, September 2013
- Switzar, Linda; van Angeren, Jordy; Pinkse, Martijn
- PROTEOMICS, Vol. 13, Issue 20
Development of an Automated, High-throughput Sample Preparation Protocol for Proteomics Analysis: Automation of Filter-aided Sample Preparation
journal, June 2015
- Arul, Albert-Baskar; Byambadorj, Munkhtsolmon; Han, Na-Young
- Bulletin of the Korean Chemical Society, Vol. 36, Issue 7
Skyline: an open source document editor for creating and analyzing targeted proteomics experiments
journal, February 2010
- MacLean, Brendan; Tomazela, Daniela M.; Shulman, Nicholas
- Bioinformatics, Vol. 26, Issue 7
Comprehensive and Scalable Highly Automated MS-Based Proteomic Workflow for Clinical Biomarker Discovery in Human Plasma
journal, July 2014
- Dayon, Loïc; Núñez Galindo, Antonio; Corthésy, John
- Journal of Proteome Research, Vol. 13, Issue 8
Protein Measurement with the Folin Phenol Reagent
journal, November 1951
- Lowry, OliverH.; Rosebrough, NiraJ.; Farr, A. Lewis
- Journal of Biological Chemistry, Vol. 193, Issue 1