Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform
Abstract
Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due to improved ionization efficiency, but the time required for sample loading, column washing, and regeneration in these systems can lead to low measurement throughput and inefficient utilization of the mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically increases the throughput of label-free SCP using two parallel subsystems to multiplex sample loading, online desalting, analysis, and column regeneration. The integration of MS1-based feature matching increased proteome coverage when short LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference in median peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An average of 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to a measurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identified nearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles.more »
- Authors:
-
- Brigham Young Univ., Provo, UT (United States)
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)
- Brigham Young Univ., Provo, UT (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)
- Publication Date:
- Research Org.:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
- Sponsoring Org.:
- USDOE; National Institute of General Medical Sciences (NIGMS)
- OSTI Identifier:
- 1983636
- Report Number(s):
- PNNL-SA-170317
Journal ID: ISSN 0003-2700
- Grant/Contract Number:
- AC05-76RL01830; R01GM138931
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Analytical Chemistry
- Additional Journal Information:
- Journal Volume: 94; Journal Issue: 15; Journal ID: ISSN 0003-2700
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; proteomics, nanoPOTS; cells; liquid chromatography; mass spectrometry; peptides and proteins; protein identification
Citation Formats
Webber, Kei G. I., Truong, Thy, Johnston, S. Madisyn, Zapata, Sebastian E., Liang, Yiran, Davis, Jacob M., Buttars, Alexander D., Smith, Fletcher B., Jones, Hailey E., Mahoney, Arianna C., Carson, Richard H., Nwosu, Andikan J., Heninger, Jacob L., Liyu, Andrey V., Nordin, Gregory P., Zhu, Ying, and Kelly, Ryan T. Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform. United States: N. p., 2022.
Web. doi:10.1021/acs.analchem.2c00646.
Webber, Kei G. I., Truong, Thy, Johnston, S. Madisyn, Zapata, Sebastian E., Liang, Yiran, Davis, Jacob M., Buttars, Alexander D., Smith, Fletcher B., Jones, Hailey E., Mahoney, Arianna C., Carson, Richard H., Nwosu, Andikan J., Heninger, Jacob L., Liyu, Andrey V., Nordin, Gregory P., Zhu, Ying, & Kelly, Ryan T. Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform. United States. https://doi.org/10.1021/acs.analchem.2c00646
Webber, Kei G. I., Truong, Thy, Johnston, S. Madisyn, Zapata, Sebastian E., Liang, Yiran, Davis, Jacob M., Buttars, Alexander D., Smith, Fletcher B., Jones, Hailey E., Mahoney, Arianna C., Carson, Richard H., Nwosu, Andikan J., Heninger, Jacob L., Liyu, Andrey V., Nordin, Gregory P., Zhu, Ying, and Kelly, Ryan T. Wed .
"Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform". United States. https://doi.org/10.1021/acs.analchem.2c00646. https://www.osti.gov/servlets/purl/1983636.
@article{osti_1983636,
title = {Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform},
author = {Webber, Kei G. I. and Truong, Thy and Johnston, S. Madisyn and Zapata, Sebastian E. and Liang, Yiran and Davis, Jacob M. and Buttars, Alexander D. and Smith, Fletcher B. and Jones, Hailey E. and Mahoney, Arianna C. and Carson, Richard H. and Nwosu, Andikan J. and Heninger, Jacob L. and Liyu, Andrey V. and Nordin, Gregory P. and Zhu, Ying and Kelly, Ryan T.},
abstractNote = {Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due to improved ionization efficiency, but the time required for sample loading, column washing, and regeneration in these systems can lead to low measurement throughput and inefficient utilization of the mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically increases the throughput of label-free SCP using two parallel subsystems to multiplex sample loading, online desalting, analysis, and column regeneration. The integration of MS1-based feature matching increased proteome coverage when short LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference in median peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An average of 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to a measurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identified nearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles. Finally, we explored the possibility of measuring cancer therapeutic targets with a pilot study comparing the K562 and Jurkat leukemia cell lines. This work demonstrates the feasibility of high-throughput label-free single-cell proteomics.},
doi = {10.1021/acs.analchem.2c00646},
journal = {Analytical Chemistry},
number = 15,
volume = 94,
place = {United States},
year = {Wed Apr 06 00:00:00 EDT 2022},
month = {Wed Apr 06 00:00:00 EDT 2022}
}
Works referenced in this record:
Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ
journal, June 2014
- Cox, Jürgen; Hein, Marco Y.; Luber, Christian A.
- Molecular & Cellular Proteomics, Vol. 13, Issue 9
Single-Cell Analysis in Biotechnology, Systems Biology, and Biocatalysis
journal, July 2012
- Fritzsch, Frederik S. O.; Dusny, Christian; Frick, Oliver
- Annual Review of Chemical and Biomolecular Engineering, Vol. 3, Issue 1
Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS
journal, June 2018
- Zhu, Ying; Clair, Geremy; Chrisler, William B.
- Angewandte Chemie, Vol. 130, Issue 38
The emerging landscape of single-molecule protein sequencing technologies
journal, June 2021
- Alfaro, Javier Antonio; Bohländer, Peggy; Dai, Mingjie
- Nature Methods, Vol. 18, Issue 6
Increased Throughput and Reduced Carryover of Mass Spectrometry-Based Proteomics Using a High-Efficiency Nonsplit Nanoflow Parallel Dual-Column Capillary HPLC System
journal, May 2008
- Wang, Hong; Hanash, Samir M.
- Journal of Proteome Research, Vol. 7, Issue 7
A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day*
journal, July 2015
- Hosp, Fabian; Scheltema, Richard A.; Eberl, H. Christian
- Molecular & Cellular Proteomics, Vol. 14, Issue 7
Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-Cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus laevis and Zebrafish Embryos
journal, February 2019
- Lombard-Banek, Camille; Moody, Sally A.; Manzini, M. Chiara
- Analytical Chemistry, Vol. 91, Issue 7
Picoelectrospray Ionization Mass Spectrometry Using Narrow-Bore Chemically Etched Emitters
journal, October 2013
- Marginean, Ioan; Tang, Keqi; Smith, Richard D.
- Journal of The American Society for Mass Spectrometry, Vol. 25, Issue 1
Ultrahigh-Throughput Proteomics Using Fast RPLC Separations with ESI-MS/MS
journal, October 2005
- Shen, Yufeng; Smith, Richard D.; Unger, Klaus K.
- Analytical Chemistry, Vol. 77, Issue 20
Single Cell Proteomics by Data-Independent Acquisition To Study Embryonic Asymmetry in Xenopus laevis
journal, June 2019
- Saha-Shah, Anumita; Esmaeili, Melody; Sidoli, Simone
- Analytical Chemistry, Vol. 91, Issue 14
Stochastic Gene Expression in a Single Cell
journal, August 2002
- Elowitz, M. B.
- Science, Vol. 297, Issue 5584
Dual LC–MS Platform for High-Throughput Proteome Analysis
journal, October 2013
- Orton, Dennis J.; Wall, Mark J.; Doucette, Alan A.
- Journal of Proteome Research, Vol. 12, Issue 12
Trapped ion mobility spectrometry and PASEF enable in-depth lipidomics from minimal sample amounts
journal, January 2020
- Vasilopoulou, Catherine G.; Sulek, Karolina; Brunner, Andreas-David
- Nature Communications, Vol. 11, Issue 1
Advances in proteomics data analysis and display using an accurate mass and time tag approach
journal, January 2006
- Zimmer, Jennifer S. D.; Monroe, Matthew E.; Qian, Wei-Jun
- Mass Spectrometry Reviews, Vol. 25, Issue 3
Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography–Mass Spectrometry for High-Throughput Single-Cell Proteomics
journal, July 2020
- Williams, Sarah M.; Liyu, Andrey V.; Tsai, Chia-Feng
- Analytical Chemistry, Vol. 92, Issue 15
Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog ( Xenopus ) Embryo
journal, January 2016
- Lombard-Banek, Camille; Moody, Sally A.; Nemes, Peter
- Angewandte Chemie International Edition, Vol. 55, Issue 7
Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering
journal, May 2022
- Woo, Jongmin; Clair, Geremy C.; Williams, Sarah M.
- Cell Systems, Vol. 13, Issue 5
Fully Automated Four-Column Capillary LC−MS System for Maximizing Throughput in Proteomic Analyses
journal, January 2008
- Livesay, Eric A.; Tang, Keqi; Taylor, Beverley K.
- Analytical Chemistry, Vol. 80, Issue 1
Single‐Cell Analysis Using Droplet Microfluidics
journal, November 2019
- Matuła, Kinga; Rivello, Francesca; Huck, Wilhelm T. S.
- Advanced Biosystems, Vol. 4, Issue 1
Defining the carrier proteome limit for single-cell proteomics
journal, December 2020
- Cheung, Tommy K.; Lee, Chien-Yun; Bayer, Florian P.
- Nature Methods, Vol. 18, Issue 1
Proteome Profiling of 1 to 5 Spiked Circulating Tumor Cells Isolated from Whole Blood Using Immunodensity Enrichment, Laser Capture Microdissection, Nanodroplet Sample Processing, and Ultrasensitive nanoLC–MS
journal, September 2018
- Zhu, Ying; Podolak, Jennifer; Zhao, Rui
- Analytical Chemistry, Vol. 90, Issue 20
A simple dual online ultra-high pressure liquid chromatography system (sDO-UHPLC) for high throughput proteome analysis
journal, January 2015
- Lee, Hangyeore; Mun, Dong-Gi; Bae, Jingi
- The Analyst, Vol. 140, Issue 16
Making Broad Proteome Protein Measurements in 1−5 min Using High-Speed RPLC Separations and High-Accuracy Mass Measurements
journal, December 2005
- Shen, Yufeng; Strittmatter, Eric F.; Zhang, Rui
- Analytical Chemistry, Vol. 77, Issue 23
Combined tissue and fluid proteomics with Tandem Mass Tags to identify low-abundance protein biomarkers of disease in peripheral body fluid: An Alzheimer's Disease case study: Combined tissue and fluid proteomics with Tandem Mass Tags
journal, December 2016
- Russell, Claire L.; Heslegrave, Amanda; Mitra, Vikram
- Rapid Communications in Mass Spectrometry, Vol. 31, Issue 2
Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells
journal, February 2018
- Zhu, Ying; Piehowski, Paul D.; Zhao, Rui
- Nature Communications, Vol. 9, Issue 1
Improved Single-Cell Proteome Coverage Using Narrow-Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry
journal, January 2020
- Cong, Yongzheng; Liang, Yiran; Motamedchaboki, Khatereh
- Analytical Chemistry, Vol. 92, Issue 3
TCRD and Pharos 2021: mining the human proteome for disease biology
journal, November 2020
- Sheils, Timothy K.; Mathias, Stephen L.; Kelleher, Keith J.
- Nucleic Acids Research, Vol. 49, Issue D1
Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell
journal, January 2021
- Cong, Yongzheng; Motamedchaboki, Khatereh; Misal, Santosh A.
- Chemical Science, Vol. 12, Issue 3
Lowering Sample Requirements to Study Tyrosine Kinase Signaling Using Phosphoproteomics with the TMT Calibrator Approach
journal, September 2020
- Fang, Bin; Izumi, Victoria; Rix, Lily L. Remsing
- PROTEOMICS, Vol. 20, Issue 24
A fully automated dual-online multifunctional ultrahigh pressure liquid chromatography system for high-throughput proteomics analysis
journal, February 2014
- Lee, Hangyeore; Lee, Jung Hwa; Kim, Hokeun
- Journal of Chromatography A, Vol. 1329
Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists
journal, November 2008
- Huang, Da Wei; Sherman, Brad T.; Lempicki, Richard A.
- Nucleic Acids Research, Vol. 37, Issue 1
Wogonin sensitizes resistant malignant cells to TNFα- and TRAIL-induced apoptosis
journal, December 2006
- Fas, Stefanie C.; Baumann, Sven; Zhu, Jia Yun
- Blood, Vol. 108, Issue 12
apQuant: Accurate Label-Free Quantification by Quality Filtering
journal, November 2018
- Doblmann, Johannes; Dusberger, Frederico; Imre, Richard
- Journal of Proteome Research
Single Cell Proteomics Using Frog ( Xenopus laevis ) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content
journal, June 2016
- Sun, Liangliang; Dubiak, Kyle M.; Peuchen, Elizabeth H.
- Analytical Chemistry, Vol. 88, Issue 13
Ultra‐high sensitivity mass spectrometry quantifies single‐cell proteome changes upon perturbation
journal, February 2022
- Brunner, Andreas‐David; Thielert, Marvin; Vasilopoulou, Catherine
- Molecular Systems Biology, Vol. 18, Issue 3
Reversal of inflammation-associated dihydrodiol dehydrogenases (AKR1C1 and AKR1C2) overexpression and drug resistance in nonsmall cell lung cancer cells by wogonin and chrysin
journal, January 2007
- Wang, Hao-Wei; Lin, Chin-Ping; Chiu, Jen-Hwey
- International Journal of Cancer, Vol. 120, Issue 9
Improved Sensitivity of Ultralow Flow LC–MS-Based Proteomic Profiling of Limited Samples Using Monolithic Capillary Columns and FAIMS Technology
journal, October 2020
- Greguš, Michal; Kostas, James C.; Ray, Somak
- Analytical Chemistry, Vol. 92, Issue 21
Streamlined single-cell proteomics by an integrated microfluidic chip and data-independent acquisition mass spectrometry
journal, January 2022
- Gebreyesus, Sofani Tafesse; Siyal, Asad Ali; Kitata, Reta Birhanu
- Nature Communications, Vol. 13, Issue 1
The MaxQuant computational platform for mass spectrometry-based shotgun proteomics
journal, October 2016
- Tyanova, Stefka; Temu, Tikira; Cox, Juergen
- Nature Protocols, Vol. 11, Issue 12
Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2
journal, January 2021
- Specht, Harrison; Emmott, Edward; Petelski, Aleksandra A.
- Genome Biology, Vol. 22, Issue 1
Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources
journal, December 2008
- Huang, Da Wei; Sherman, Brad T.; Lempicki, Richard A.
- Nature Protocols, Vol. 4, Issue 1
Chemical and biological single cell analysis
journal, February 2010
- Schmid, Andreas; Kortmann, Hendrik; Dittrich, Petra S.
- Current Opinion in Biotechnology, Vol. 21, Issue 1
Single-cell Proteomics: Progress and Prospects
journal, November 2020
- Kelly, Ryan T.
- Molecular & Cellular Proteomics, Vol. 19, Issue 11
High-Throughput, Comprehensive Single-Cell Proteomic Analysis of Xenopus laevis Embryos at the 50-Cell Stage Using a Microplate-Based MICROFASP System
journal, February 2022
- Zhang, Zhenbin; Dubiak, Kyle M.; Shishkova, Evgenia
- Analytical Chemistry, Vol. 94, Issue 7