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

Yuqian Gao; Thomas L. Fillmore; Nathalie Munoz; Gayle J. Bentley; Christopher W. Johnson; Joonhoon Kim; Jamie A. Meadows; Jeremy D. Zucker; Meagan C. Burnet; Anna K. Lipton; Aivett Bilbao; Daniel J. Orton; Young Mo Kim; Ronald J. Moore; Errol W. Robinson; Scott E. Baker; Bobbie Jo M. Webb-Robertson; Adam M. Guss; John M. Gladden; Gregg T. Beckham; Jon K. Magnuson; Kristin E. Burnum-Johnson

doi:10.3389/fbioe.2020.603488

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

Yuqian Gao, Thomas L. Fillmore, Nathalie Munoz, Gayle J. Bentley, Christopher W. Johnson, Joonhoon Kim, Jamie A. Meadows, Jeremy D. Zucker, Meagan C. Burnet, Anna K. Lipton, Aivett Bilbao, Daniel J. Orton, Young Mo Kim, Ronald J. Moore, Errol W. Robinson, Scott E. Baker, Bobbie Jo M. Webb-Robertson, Adam M. Guss, John M. Gladden, Gregg T. BeckhamJon K. Magnuson, Kristin E. Burnum-Johnson

Synthetic Biology

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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.

Original language	English
Article number	603488
Journal	Frontiers in Bioengineering and Biotechnology
Volume	8
DOIs	https://doi.org/10.3389/fbioe.2020.603488
State	Published - Dec 2 2020

Funding

A portion of this research was performed at Pacific Northwest National Laboratory (PNNL) using EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. PNNL is a multiprogram national laboratory operated by Battelle for the Department of Energy (DOE) under Contract DE-AC05-76RLO 1830. This work was authored in part by Alliance for Sustainable Energy, LLC, the manager and operator of the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. Funding was provided by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office (BETO) for the Agile BioFoundry (under Award No. DE-NL0030038). The views expressed in the article do not necessarily represent the views of the U.S. Department of Energy or the United States Government.

Funders	Funder number
U.S. Department of Energy Office of Energy Efficiency and Renewable Energy BioEnergy Technologies Office
U.S. Department of Energy	DE-AC36-08GO28308, DE-AC05-76RLO 1830
Office of Science
Biological and Environmental Research
Oak Ridge National Laboratory	DE-AC05-00OR22725
National Renewable Energy Laboratory
Pacific Northwest National Laboratory
Bioenergy Technologies Office
Agile BioFoundry	DE-NL0030038

Keywords

Pseudomonas putida KT2440
central carbon metabolism
mass spectrometry
selected reaction monitoring
targeted proteomics

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10.3389/fbioe.2020.603488

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Gao, Y., Fillmore, T. L., Munoz, N., Bentley, G. J., Johnson, C. W., Kim, J., Meadows, J. A., Zucker, J. D., Burnet, M. C., Lipton, A. K., Bilbao, A., Orton, D. J., Kim, Y. M., Moore, R. J., Robinson, E. W., Baker, S. E., Webb-Robertson, B. J. M., Guss, A. M., Gladden, J. M., ... Burnum-Johnson, K. E. (2020). High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440. Frontiers in Bioengineering and Biotechnology, 8, Article 603488. https://doi.org/10.3389/fbioe.2020.603488

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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.",

keywords = "Pseudomonas putida KT2440, central carbon metabolism, mass spectrometry, selected reaction monitoring, targeted proteomics",

author = "Yuqian Gao and Fillmore, {Thomas L.} and Nathalie Munoz and Bentley, {Gayle J.} and Johnson, {Christopher W.} and Joonhoon Kim and Meadows, {Jamie A.} and Zucker, {Jeremy D.} and Burnet, {Meagan C.} and Lipton, {Anna K.} and Aivett Bilbao 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 M.} and Gladden, {John M.} and Beckham, {Gregg T.} and Magnuson, {Jon K.} and Burnum-Johnson, {Kristin E.}",

note = "Publisher Copyright: {\textcopyright} Copyright {\textcopyright} 2020 Gao, Fillmore, Munoz, Bentley, Johnson, Kim, Meadows, Zucker, Burnet, Lipton, Bilbao, Orton, Kim, Moore, Robinson, Baker, Webb-Robertson, Guss, Gladden, Beckham, Magnuson and Burnum-Johnson.",

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Gao, Y, Fillmore, TL, Munoz, N, Bentley, GJ, Johnson, CW, Kim, J, Meadows, JA, Zucker, JD, Burnet, MC, Lipton, AK, Bilbao, A, Orton, DJ, Kim, YM, Moore, RJ, Robinson, EW, Baker, SE, Webb-Robertson, BJM, Guss, AM, Gladden, JM, Beckham, GT, Magnuson, JK & Burnum-Johnson, KE 2020, 'High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440', Frontiers in Bioengineering and Biotechnology, vol. 8, 603488. https://doi.org/10.3389/fbioe.2020.603488

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AU - Meadows, Jamie A.

AU - Zucker, Jeremy D.

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AU - Lipton, Anna K.

AU - Bilbao, Aivett

AU - Orton, Daniel J.

AU - Kim, Young Mo

AU - Moore, Ronald J.

AU - Robinson, Errol W.

AU - Baker, Scott E.

AU - Webb-Robertson, Bobbie Jo M.

AU - Guss, Adam M.

AU - Gladden, John M.

AU - Beckham, Gregg T.

AU - Magnuson, Jon K.

AU - Burnum-Johnson, Kristin E.

N1 - Publisher Copyright: © Copyright © 2020 Gao, Fillmore, Munoz, Bentley, Johnson, Kim, Meadows, Zucker, Burnet, Lipton, Bilbao, Orton, Kim, Moore, Robinson, Baker, Webb-Robertson, Guss, Gladden, Beckham, Magnuson and Burnum-Johnson.

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N2 - 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.

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

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