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Title: High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440

Abstract

Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.

Authors:
 [1];  [2];  [3];  [4];  [4]; ORCiD logo [3];  [5]; ORCiD logo [3];  [3];  [3]; ORCiD logo [3]; ORCiD logo [2]; ORCiD logo [3];  [2];  [3]; ORCiD logo [3];  [3];  [1];  [5];  [4] more »; ORCiD logo [3]; ORCiD logo [3] « less
  1. Agile BioFoundry, Emeryville, CA (United States)
  2. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  3. Agile BioFoundry, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  4. Agile BioFoundry, Emeryville, CA (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States)
  5. Agile BioFoundry, Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Transportation Office. Bioenergy Technologies Office
OSTI Identifier:
1728466
Alternate Identifier(s):
OSTI ID: 1760672; OSTI ID: 1813134
Report Number(s):
PNNL-SA-156111; NREL/JA-2A00-78200
Journal ID: ISSN 2296-4185
Grant/Contract Number:  
AC05-76RL01830; AC36-08GO28308; AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Frontiers in Bioengineering and Biotechnology
Additional Journal Information:
Journal Volume: 8; Journal ID: ISSN 2296-4185
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; targeted proteomics; Pseudomonas putida KT2440; mass spectrometry; Selected reaction monitoring; central carbon metablolism; selected 18 reaction monitoring (SRM); central carbon metabolism

Citation Formats

Gao, Yuqian, Fillmore, Thomas L., Munoz Munoz, Nathalie, Bentley, Gayle, Johnson, Christopher W., Kim, Joonhoon, Meadows, Jamie, Zucker, Jeremy D., Burnet, Meagan C., Lipton, Anna K., Bilbao Pena, Aivett, Orton, Daniel J., Kim, Young-Mo, Moore, Ronald J., Robinson, Errol W., Baker, Scott E., Webb-Robertson, Bobbie-Jo M., Guss, Adam, Gladden, John M., Beckham, Gregg T., Magnuson, Jon K., and Burnum-Johnson, Kristin E.. High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440. United States: N. p., 2020. Web. https://doi.org/10.3389/fbioe.2020.603488.
Gao, Yuqian, Fillmore, Thomas L., Munoz Munoz, Nathalie, Bentley, Gayle, Johnson, Christopher W., Kim, Joonhoon, Meadows, Jamie, Zucker, Jeremy D., Burnet, Meagan C., Lipton, Anna K., Bilbao Pena, Aivett, Orton, Daniel J., Kim, Young-Mo, Moore, Ronald J., Robinson, Errol W., Baker, Scott E., Webb-Robertson, Bobbie-Jo M., Guss, Adam, Gladden, John M., Beckham, Gregg T., Magnuson, Jon K., & Burnum-Johnson, Kristin E.. High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440. United States. https://doi.org/10.3389/fbioe.2020.603488
Gao, Yuqian, Fillmore, Thomas L., Munoz Munoz, Nathalie, Bentley, Gayle, Johnson, Christopher W., Kim, Joonhoon, Meadows, Jamie, Zucker, Jeremy D., Burnet, Meagan C., Lipton, Anna K., Bilbao Pena, Aivett, Orton, Daniel J., Kim, Young-Mo, Moore, Ronald J., Robinson, Errol W., Baker, Scott E., Webb-Robertson, Bobbie-Jo M., Guss, Adam, Gladden, John M., Beckham, Gregg T., Magnuson, Jon K., and Burnum-Johnson, Kristin E.. Wed . "High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440". United States. https://doi.org/10.3389/fbioe.2020.603488. https://www.osti.gov/servlets/purl/1728466.
@article{osti_1728466,
title = {High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440},
author = {Gao, Yuqian and Fillmore, Thomas L. and Munoz Munoz, Nathalie and Bentley, Gayle and Johnson, Christopher W. and Kim, Joonhoon and Meadows, Jamie and Zucker, Jeremy D. and Burnet, Meagan C. and Lipton, Anna K. and Bilbao Pena, Aivett and Orton, Daniel J. and Kim, Young-Mo and Moore, Ronald J. and Robinson, Errol W. and Baker, Scott E. and Webb-Robertson, Bobbie-Jo M. and Guss, Adam and Gladden, John M. and Beckham, Gregg T. and Magnuson, Jon K. and Burnum-Johnson, Kristin E.},
abstractNote = {Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.},
doi = {10.3389/fbioe.2020.603488},
journal = {Frontiers in Bioengineering and Biotechnology},
number = ,
volume = 8,
place = {United States},
year = {2020},
month = {12}
}

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