High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip
Abstract
Abstract Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.
- Publication Date:
- Research Org.:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Org.:
- USDO; USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)
- OSTI Identifier:
- 1828225
- Alternate Identifier(s):
- OSTI ID: 1831467
- Report Number(s):
- PNNL-SA-159977
Journal ID: ISSN 2041-1723; 6246; PII: 26514
- Grant/Contract Number:
- AC05-76RL01830; U01HL122703; U01HL148860; P41GM103493
- Resource Type:
- Published Article
- Journal Name:
- Nature Communications
- Additional Journal Information:
- Journal Name: Nature Communications Journal Volume: 12 Journal Issue: 1; Journal ID: ISSN 2041-1723
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United Kingdom
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; lab-on-a-chip; mass spectrometry; proteome; proteomics
Citation Formats
Woo, Jongmin, Williams, Sarah M., Markillie, Lye Meng, Feng, Song, Tsai, Chia-Feng, Aguilera-Vazquez, Victor, Sontag, Ryan L., Moore, Ronald J., Hu, Dehong, Mehta, Hardeep S., Cantlon-Bruce, Joshua, Liu, Tao, Adkins, Joshua N., Smith, Richard D., Clair, Geremy C., Pasa-Tolic, Ljiljana, and Zhu, Ying. High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip. United Kingdom: N. p., 2021.
Web. doi:10.1038/s41467-021-26514-2.
Woo, Jongmin, Williams, Sarah M., Markillie, Lye Meng, Feng, Song, Tsai, Chia-Feng, Aguilera-Vazquez, Victor, Sontag, Ryan L., Moore, Ronald J., Hu, Dehong, Mehta, Hardeep S., Cantlon-Bruce, Joshua, Liu, Tao, Adkins, Joshua N., Smith, Richard D., Clair, Geremy C., Pasa-Tolic, Ljiljana, & Zhu, Ying. High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip. United Kingdom. https://doi.org/10.1038/s41467-021-26514-2
Woo, Jongmin, Williams, Sarah M., Markillie, Lye Meng, Feng, Song, Tsai, Chia-Feng, Aguilera-Vazquez, Victor, Sontag, Ryan L., Moore, Ronald J., Hu, Dehong, Mehta, Hardeep S., Cantlon-Bruce, Joshua, Liu, Tao, Adkins, Joshua N., Smith, Richard D., Clair, Geremy C., Pasa-Tolic, Ljiljana, and Zhu, Ying. Fri .
"High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip". United Kingdom. https://doi.org/10.1038/s41467-021-26514-2.
@article{osti_1828225,
title = {High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip},
author = {Woo, Jongmin and Williams, Sarah M. and Markillie, Lye Meng and Feng, Song and Tsai, Chia-Feng and Aguilera-Vazquez, Victor and Sontag, Ryan L. and Moore, Ronald J. and Hu, Dehong and Mehta, Hardeep S. and Cantlon-Bruce, Joshua and Liu, Tao and Adkins, Joshua N. and Smith, Richard D. and Clair, Geremy C. and Pasa-Tolic, Ljiljana and Zhu, Ying},
abstractNote = {Abstract Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.},
doi = {10.1038/s41467-021-26514-2},
journal = {Nature Communications},
number = 1,
volume = 12,
place = {United Kingdom},
year = {2021},
month = {10}
}
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Works referenced in this record:
Single-cell analysis targeting the proteome
journal, February 2020
- Labib, Mahmoud; Kelley, Shana O.
- Nature Reviews Chemistry, Vol. 4, Issue 3
Picoflow Liquid Chromatography–Mass Spectrometry for Ultrasensitive Bottom-Up Proteomics Using 2-μm-i.d. Open Tubular Columns
journal, March 2020
- Xiang, Piliang; Zhu, Ying; Yang, Yu
- Analytical Chemistry, Vol. 92, Issue 7
The sva package for removing batch effects and other unwanted variation in high-throughput experiments
journal, January 2012
- Leek, Jeffrey T.; Johnson, W. Evan; Parker, Hilary S.
- Bioinformatics, Vol. 28, Issue 6
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
CD68/macrosialin: not just a histochemical marker
journal, November 2016
- Chistiakov, Dimitry A.; Killingsworth, Murry C.; Myasoedova, Veronika A.
- Laboratory Investigation, Vol. 97, Issue 1
Fully Automated Sample Processing and Analysis Workflow for Low-Input Proteome Profiling
journal, December 2020
- Liang, Yiran; Acor, Hayden; McCown, Michaela A.
- Analytical Chemistry, Vol. 93, Issue 3
Expression of macrophage-selective markers in human and rodent adipocytes
journal, September 2005
- Khazen, Wael; M'Bika, Jean-Pierre; Tomkiewicz, Céline
- FEBS Letters, Vol. 579, Issue 25
Junctional Adhesion Molecule 1 Regulates Epithelial Cell Morphology through Effects on β1 Integrins and Rap1 Activity
journal, March 2005
- Mandell, Kenneth J.; Babbin, Brian A.; Nusrat, Asma
- Journal of Biological Chemistry, Vol. 280, Issue 12
Mass-spectrometric exploration of proteome structure and function
journal, September 2016
- Aebersold, Ruedi; Mann, Matthias
- Nature, Vol. 537, Issue 7620
Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer
journal, July 2016
- Zhang, Hui; Liu, Tao; Zhang, Zhen
- Cell, Vol. 166, Issue 3
MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics
journal, April 2017
- Kong, Andy T.; Leprevost, Felipe V.; Avtonomov, Dmitry M.
- Nature Methods, Vol. 14, Issue 5
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
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 International Edition, Vol. 57, Issue 38
The Perseus computational platform for comprehensive analysis of (prote)omics data
journal, June 2016
- Tyanova, Stefka; Temu, Tikira; Sinitcyn, Pavel
- Nature Methods, Vol. 13, Issue 9
Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling
journal, October 2018
- Shao, Xi; Wang, Xuantang; Guan, Sheng
- Analytical Chemistry, Vol. 90, Issue 23
An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics
journal, May 2020
- Tsai, Chia-Feng; Zhao, Rui; Williams, Sarah M.
- Molecular & Cellular Proteomics, Vol. 19, Issue 5
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
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
High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform
journal, September 2019
- Dou, Maowei; Clair, Geremy; Tsai, Chia-Feng
- Analytical Chemistry, Vol. 91, Issue 20
Quantitative single-cell proteomics as a tool to characterize cellular hierarchies
journal, June 2021
- Schoof, Erwin M.; Furtwängler, Benjamin; Üresin, Nil
- Nature Communications, Vol. 12, Issue 1
Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma
journal, February 2019
- Jia, Man; Yan, Xiaoyi; Jiang, Xinyu
- American Journal of Respiratory and Critical Care Medicine, Vol. 199, Issue 4
Spatial proteomics: a powerful discovery tool for cell biology
journal, January 2019
- Lundberg, Emma; Borner, Georg H. H.
- Nature Reviews Molecular Cell Biology, Vol. 20, Issue 5
Soluble polysialylated NCAM: a novel player of the innate immune system in the lung
journal, April 2013
- Ulm, Christina; Saffarzadeh, Mona; Mahavadi, Poornima
- Cellular and Molecular Life Sciences, Vol. 70, Issue 19
Droplet-Based Multivolume Digital Polymerase Chain Reaction by a Surface-Assisted Multifactor Fluid Segmentation Approach
journal, December 2016
- Liu, Wen-Wen; Zhu, Ying; Feng, Yi-Ming
- Analytical Chemistry, Vol. 89, Issue 1
Cells Respond to Distinct Nanoparticle Properties with Multiple Strategies As Revealed by Single-Cell RNA-Seq
journal, October 2016
- Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.
- ACS Nano, Vol. 10, Issue 11
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
Tissue-based map of the human proteome
journal, January 2015
- Uhlen, M.; Fagerberg, L.; Hallstrom, B. M.
- Science, Vol. 347, Issue 6220
SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation
journal, October 2018
- Budnik, Bogdan; Levy, Ezra; Harmange, Guillaume
- Genome Biology, Vol. 19, Issue 1
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
Control of endothelial cell proliferation and migration by VEGF signaling to histone deacetylase 7
journal, May 2008
- Wang, S.; Li, X.; Parra, M.
- Proceedings of the National Academy of Sciences, Vol. 105, Issue 22
Measuring Signaling and RNA-Seq in the Same Cell Links Gene Expression to Dynamic Patterns of NF-κB Activation
journal, April 2017
- Lane, Keara; Van Valen, David; DeFelice, Mialy M.
- Cell Systems, Vol. 4, Issue 4
Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis
journal, March 2018
- Li, Zi-Yi; Huang, Min; Wang, Xiu-Kun
- Analytical Chemistry, Vol. 90, Issue 8
A Defective Pentose Phosphate Pathway Reduces Inflammatory Macrophage Responses during Hypercholesterolemia
journal, November 2018
- Baardman, Jeroen; Verberk, Sanne G. S.; Prange, Koen H. M.
- Cell Reports, Vol. 25, Issue 8
The impact of amplification on differential expression analyses by RNA-seq
journal, May 2016
- Parekh, Swati; Ziegenhain, Christoph; Vieth, Beate
- Scientific Reports, Vol. 6, Issue 1
Manipulating Femtoliter to Picoliter Droplets by Pins for Single Cell Analysis and Quantitative Biological Assay
journal, April 2018
- Guo, Xiao-Li; Wei, Yan; Lou, Qi
- Analytical Chemistry, Vol. 90, Issue 9
In vivo expression profile of the antiviral restriction factor and tumor-targeting antigen CD317/BST-2/HM1.24/tetherin in humans
journal, August 2011
- Erikson, E.; Adam, T.; Schmidt, S.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 33
Single-cell Proteomics: Progress and Prospects
journal, November 2020
- Kelly, Ryan T.
- Molecular & Cellular Proteomics, Vol. 19, Issue 11
Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells
journal, January 2016
- Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana
- Cell Reports, Vol. 14, Issue 2