Cell-Type-Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform
Abstract
Abstract Plant organs and tissues contain multiple cell types, which are well organized in 3‐dimensional structure to efficiently perform physiological functions such as homeostasis and response to environmental perturbation and pathogen infection. It is critically important to perform molecular measurements at the cell‐type‐specific level to discover mechanisms and unique features of cell populations that govern differentiation and respond to external perturbations. Although mass spectrometry−based proteomics has been demonstrated as an enabling discovery tool for studying plant physiology, conventional approaches require millions of cells to generate robust biological conclusions. Such requirements mask the cell‐to‐cell heterogeneities and limit the comprehensive profiling of plant proteins at spatially resolved and cell‐type‐specific resolutions. This article describes a recently developed proteomics workflow for studying a small number of plant cells by integrating laser capture microdissection, microfluidic nanodroplet−based sample preparation, and ultrasensitive liquid chromatography−mass spectrometry. Using poplar as a model tree species, we provide detailed protocols, including plant leaf and root tissue harvest, sample preparation, cryosectioning, laser microdissection, protein digestion, mass spectrometry measurement, and data analysis. We show that the workflow enables the precise identification and quantification of thousands of proteins from hundreds of isolated plant root and leaf cells. © 2021 Wiley Periodicals LLC. Basic Protocolmore »
- Authors:
-
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Brigham Young Univ., Provo, UT (United States)
- Univ. of California, Davis, CA (United States)
- Univ. of Tennessee, Knoxville, TN (United States). Center for Agricultural Synthetic Biology
- Publication Date:
- Research Org.:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1811677
- Alternate Identifier(s):
- OSTI ID: 1785042
- Report Number(s):
- PNNL-SA-160346
Journal ID: ISSN 2691-1299
- Grant/Contract Number:
- AC05-76RL01830; SC0018347
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Current Protocols
- Additional Journal Information:
- Journal Volume: 1; Journal Issue: 5; Journal ID: ISSN 2691-1299
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Cell-type-specific proteomics; NanoPOTS; Laser capture microdissection; Mass spectrometry; Poplar
Citation Formats
Balasubramanian, Vimal K., Purvine, Samuel O., Liang, Yiran, Kelly, Ryan T., Pasa‐Tolic, Ljiljana, Chrisler, William B., Blumwald, Eduardo, Stewart, C. Neal, Zhu, Ying, and Ahkami, Amir H. Cell-Type-Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform. United States: N. p., 2021.
Web. doi:10.1002/cpz1.153.
Balasubramanian, Vimal K., Purvine, Samuel O., Liang, Yiran, Kelly, Ryan T., Pasa‐Tolic, Ljiljana, Chrisler, William B., Blumwald, Eduardo, Stewart, C. Neal, Zhu, Ying, & Ahkami, Amir H. Cell-Type-Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform. United States. https://doi.org/10.1002/cpz1.153
Balasubramanian, Vimal K., Purvine, Samuel O., Liang, Yiran, Kelly, Ryan T., Pasa‐Tolic, Ljiljana, Chrisler, William B., Blumwald, Eduardo, Stewart, C. Neal, Zhu, Ying, and Ahkami, Amir H. Thu .
"Cell-Type-Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform". United States. https://doi.org/10.1002/cpz1.153. https://www.osti.gov/servlets/purl/1811677.
@article{osti_1811677,
title = {Cell-Type-Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform},
author = {Balasubramanian, Vimal K. and Purvine, Samuel O. and Liang, Yiran and Kelly, Ryan T. and Pasa‐Tolic, Ljiljana and Chrisler, William B. and Blumwald, Eduardo and Stewart, C. Neal and Zhu, Ying and Ahkami, Amir H.},
abstractNote = {Abstract Plant organs and tissues contain multiple cell types, which are well organized in 3‐dimensional structure to efficiently perform physiological functions such as homeostasis and response to environmental perturbation and pathogen infection. It is critically important to perform molecular measurements at the cell‐type‐specific level to discover mechanisms and unique features of cell populations that govern differentiation and respond to external perturbations. Although mass spectrometry−based proteomics has been demonstrated as an enabling discovery tool for studying plant physiology, conventional approaches require millions of cells to generate robust biological conclusions. Such requirements mask the cell‐to‐cell heterogeneities and limit the comprehensive profiling of plant proteins at spatially resolved and cell‐type‐specific resolutions. This article describes a recently developed proteomics workflow for studying a small number of plant cells by integrating laser capture microdissection, microfluidic nanodroplet−based sample preparation, and ultrasensitive liquid chromatography−mass spectrometry. Using poplar as a model tree species, we provide detailed protocols, including plant leaf and root tissue harvest, sample preparation, cryosectioning, laser microdissection, protein digestion, mass spectrometry measurement, and data analysis. We show that the workflow enables the precise identification and quantification of thousands of proteins from hundreds of isolated plant root and leaf cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1 : Plant tissue fixation and embedding Support Protocol 1 : Preparation of 2.5% CMC solution Support Protocol 2 : Slow freezing of CMC blocks to avoid crack development in the block Basic Protocol 2 : Preparation of cryosections Alternate Protocol : Using a vacuum manifold to dehydrate the cryosection slides (primarily for root tissues) Basic Protocol 3 : Laser capture microdissection of specific types of plant cells Basic Protocol 4 : Nanodroplet‐based sample preparation for ultrasensitive proteomic analysis Support Protocol 3 : Fabrication of nanowell chips Basic Protocol 5 : Liquid chromatography and mass spectrometry},
doi = {10.1002/cpz1.153},
journal = {Current Protocols},
number = 5,
volume = 1,
place = {United States},
year = {Thu May 27 00:00:00 EDT 2021},
month = {Thu May 27 00:00:00 EDT 2021}
}
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Works referenced in this record:
Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots
journal, June 2016
- Zhu, Yingde; Li, Hui; Bhatti, Sarabjit
- Horticulture Research, Vol. 3, Issue 1
Whole-genome draft assembly of Populus tremula x P. alba clone INRA 717-1B4
journal, December 2016
- Mader, Malte; Le Paslier, Marie-Christine; Bounon, Rémi
- Silvae Genetica, Vol. 65, Issue 2
Evaluation of two-dimensional electrophoresis and liquid chromatography - tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples
journal, July 2005
- Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick
- ELECTROPHORESIS, Vol. 26, Issue 14
Comparative Proteomic Analysis of Arabidopsis Mature Pollen and Germinated Pollen
journal, May 2009
- Zou, Junjie; Song, Lianfen; Zhang, Wenzheng
- Journal of Integrative Plant Biology, Vol. 51, Issue 5
Genome Sequences of Populus tremula Chloroplast and Mitochondrion: Implications for Holistic Poplar Breeding
journal, January 2016
- Kersten, Birgit; Faivre Rampant, Patricia; Mader, Malte
- PLOS ONE, Vol. 11, Issue 1
Transcriptomic and functional analysis of cucumber ( Cucumis sativus L.) fruit phloem during early development
journal, October 2018
- Sui, Xiaolei; Nie, Jing; Li, Xin
- The Plant Journal, Vol. 96, Issue 5
DAnTE: a statistical tool for quantitative analysis of -omics data
journal, May 2008
- Polpitiya, A. D.; Qian, W. -J.; Jaitly, N.
- Bioinformatics, Vol. 24, Issue 13
LASER MICRODISSECTION OF PLANT TISSUE: What You See Is What You Get
journal, June 2006
- Nelson, Timothy; Tausta, S. Lori; Gandotra, Neeru
- Annual Review of Plant Biology, Vol. 57, Issue 1
Proteomics: a powerful tool to study plant responses to biotic stress
journal, November 2019
- Liu, Yahui; Lu, Song; Liu, Kefu
- Plant Methods, Vol. 15, Issue 1
Unique Physiological and Transcriptional Shifts under Combinations of Salinity, Drought, and Heat
journal, March 2017
- Shaar-Moshe, Lidor; Blumwald, Eduardo; Peleg, Zvi
- Plant Physiology, Vol. 174, Issue 1
Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling
journal, November 2016
- Martin, Laetitia B. B.; Nicolas, Philippe; Matas, Antonio J.
- Nature Protocols, Vol. 11, Issue 12
The protein expression landscape of the Arabidopsis root
journal, March 2012
- Petricka, J. J.; Schauer, M. A.; Megraw, M.
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 18
Plant Systems Biology at the Single-Cell Level
journal, November 2017
- Libault, Marc; Pingault, Lise; Zogli, Prince
- Trends in Plant Science, Vol. 22, Issue 11
A Two-dimensional Electrophoresis Proteomic Reference Map and Systematic Identification of 1367 Proteins from a Cell Suspension Culture of the Model Legume Medicago truncatula
journal, November 2005
- Lei, Zhentian; Elmer, Aaron M.; Watson, Bonnie S.
- Molecular & Cellular Proteomics, Vol. 4, Issue 11
Spatially Resolved Proteome Profiling of <200 Cells from Tomato Fruit Pericarp by Integrating Laser-Capture Microdissection with Nanodroplet Sample Preparation
journal, August 2018
- Liang, Yiran; Zhu, Ying; Dou, Maowei
- Analytical Chemistry, Vol. 90, Issue 18
Unravelling the proteomic profile of rice meiocytes during early meiosis
journal, July 2014
- Collado-Romero, Melania; Alós, Enriqueta; Prieto, Pilar
- Frontiers in Plant Science, Vol. 5
Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks
journal, January 2012
- Krasensky, J.; Jonak, C.
- Journal of Experimental Botany, Vol. 63, Issue 4
Biological significance of RNA-seq and single-cell genomic research in woody plants
journal, April 2019
- Tang, Wei; Tang, Anna Y.
- Journal of Forestry Research, Vol. 30, Issue 5
Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response
journal, January 2011
- Van Cutsem, Emmanuel; Simonart, Géraldine; Degand, Hervé
- PROTEOMICS, Vol. 11, Issue 3
Quantitative transcriptomic analysis of abscisic acid-induced and reactive oxygen species-dependent expression changes and proteomic profiling in Arabidopsis suspension cells: ABA- and ROS-regulated expression changes
journal, April 2011
- Böhmer, Maik; Schroeder, Julian I.
- The Plant Journal, Vol. 67, 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
A method for obtaining high integrity RNA from developing aleurone cells and starchy endosperm in rice (Oryza sativa L.) by laser microdissection
journal, September 2007
- Ishimaru, Tsutomu; Nakazono, Mikio; Masumura, Takehiro
- Plant Science, Vol. 173, Issue 3
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
Tissue proteomics using chemical immobilization and mass spectrometry
journal, January 2015
- Shah, Punit; Zhang, Bai; Choi, Caitlin
- Analytical Biochemistry, Vol. 469
Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples
journal, June 2019
- Dou, Maowei; Tsai, Chia-Feng; Piehowski, Paul D.
- Analytical Chemistry, Vol. 91, Issue 15
Plant proteomics: Concepts, applications, and novel strategies for data interpretation
journal, July 2008
- Baginsky, Sacha
- Mass Spectrometry Reviews, Vol. 28, Issue 1
Label-Free Comparative Proteomic Analysis Combined with Laser-Capture Microdissection Suggests Important Roles of Stress Responses in the Black Layer of Maize Kernels
journal, February 2020
- Chen, Quanquan; Huang, Ran; Xu, Zhenxiang
- International Journal of Molecular Sciences, Vol. 21, Issue 4
Conserved and Unique Features of the Maize ( Zea mays L.) Root Hair Proteome
journal, May 2011
- Nestler, Josefine; Schütz, Wolfgang; Hochholdinger, Frank
- Journal of Proteome Research, Vol. 10, Issue 5
The Al-induced proteomes of epidermal and outer cortical cells in root apex of cherry tomato ‘LA 2710’
journal, January 2020
- Yang, Shaolan; Li, Hui; Bhatti, Sarabjit
- Journal of Proteomics, Vol. 211
A cross-platform toolkit for mass spectrometry and proteomics
journal, October 2012
- Chambers, Matthew C.; Maclean, Brendan; Burke, Robert
- Nature Biotechnology, Vol. 30, Issue 10
Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics
journal, April 2010
- Schilmiller, Anthony L.; Miner, Dennis P.; Larson, Matthew
- Plant Physiology, Vol. 153, Issue 3
Unpicking the proteome in single cells
journal, January 2020
- Slavov, Nikolai
- Science, Vol. 367, Issue 6477
MASIC: A software program for fast quantitation and flexible visualization of chromatographic profiles from detected LC–MS(/MS) features
journal, June 2008
- Monroe, Matthew E.; Shaw, Jason L.; Daly, Don S.
- Computational Biology and Chemistry, Vol. 32, Issue 3
Water-deficit stress-induced anatomical changes in higher plants
journal, March 2008
- Shao, Hong-Bo; Chu, Li-Ye; Jaleel, Cheruth Abdul
- Comptes Rendus Biologies, Vol. 331, Issue 3
The Genome of Black Cottonwood, Populus trichocarpa (Torr. & Gray)
journal, September 2006
- Tuskan, G. A.; DiFazio, S.; Jansson, S.
- Science, Vol. 313, Issue 5793, p. 1596-1604
Paraffin Embedding Contributes to RNA Aggregation, Reduced RNA Yield, and Low RNA Quality
journal, November 2011
- Evers, David L.; He, Junkun; Kim, Yeon Ho
- The Journal of Molecular Diagnostics, Vol. 13, Issue 6
Single-Cell RNA Sequencing Resolves Molecular Relationships Among Individual Plant Cells
journal, February 2019
- Ryu, Kook Hui; Huang, Ling; Kang, Hyun Min
- Plant Physiology, Vol. 179, Issue 4
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
Advances in microscale separations towards nanoproteomics applications
journal, November 2017
- Yi, Lian; Piehowski, Paul D.; Shi, Tujin
- Journal of Chromatography A, Vol. 1523
Organ-specific proteome analysis for identification of abiotic stress response mechanism in crop
journal, January 2013
- Komatsu, Setsuko; Hossain, Zahed
- Frontiers in Plant Science, Vol. 4
Transcriptomic and Proteomic Analyses of Pericycle Cells of the Maize Primary Root
journal, August 2007
- Dembinsky, Diana; Woll, Katrin; Saleem, Muhammad
- Plant Physiology, Vol. 145, Issue 3
Comparative Proteomic Analysis of Cultured Suspension Cells of the Halophyte Halogeton glomeratus by iTRAQ Provides Insights into Response Mechanisms to Salt Stress
journal, February 2016
- Wang, Juncheng; Yao, Lirong; Li, Baochun
- Frontiers in Plant Science, Vol. 7
Target-Decoy Search Strategy for Mass Spectrometry-Based Proteomics
book, December 2009
- Elias, Joshua E.; Gygi, Steven P.
- Methods in Molecular Biology
Spatially Resolved Proteome Mapping of Laser Capture Microdissected Tissue with Automated Sample Transfer to Nanodroplets
journal, June 2018
- Zhu, Ying; Dou, Maowei; Piehowski, Paul D.
- Molecular & Cellular Proteomics, Vol. 17, Issue 9
A Gene Expression Map of the Arabidopsis Root
journal, December 2003
- Birnbaum, K.
- Science, Vol. 302, Issue 5652
Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics
journal, April 2009
- Zhu, Mengmeng; Dai, Shaojun; McClung, Scott
- Molecular & Cellular Proteomics, Vol. 8, Issue 4
Proteomic analyses ofOryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth
journal, April 2006
- Dai, Shaojun; Li, Lei; Chen, Taotao
- PROTEOMICS, Vol. 6, Issue 8
Universal Sample Preparation Unlocking Multimodal Molecular Tissue Imaging
journal, June 2020
- Dannhorn, Andreas; Kazanc, Emine; Ling, Stephanie
- Analytical Chemistry, Vol. 92, Issue 16
Single-cell-type Proteomics: Toward a Holistic Understanding of Plant Function
journal, September 2012
- Dai, Shaojun; Chen, Sixue
- Molecular & Cellular Proteomics, Vol. 11, Issue 12
Tissue- and Cell-Type Specific Transcriptome Profiling of Expanding Tomato Fruit Provides Insights into Metabolic and Regulatory Specialization and Cuticle Formation
journal, November 2011
- Matas, Antonio J.; Yeats, Trevor H.; Buda, Gregory J.
- The Plant Cell, Vol. 23, Issue 11
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
Impact of Post-Translational Modifications of Crop Proteins under Abiotic Stress
journal, December 2016
- Hashiguchi, Akiko; Komatsu, Setsuko
- Proteomes, Vol. 4, Issue 4
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
Proteomic analysis of tomato (Lycopersicon esculentum) pollen
journal, September 2007
- Sheoran, I. S.; Ross, A. R. S.; Olson, D. J. H.
- Journal of Experimental Botany, Vol. 58, Issue 13
Advances in plant proteomics toward improvement of crop productivity and stress resistancex
journal, April 2015
- Hu, Junjie; Rampitsch, Christof; Bykova, Natalia V.
- Frontiers in Plant Science, Vol. 6
Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures.
journal, October 1985
- Solomon, M. J.; Varshavsky, A.
- Proceedings of the National Academy of Sciences, Vol. 82, Issue 19
Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots
journal, June 2017
- Marion, Jessica; Le Bars, Romain; Satiat-Jeunemaitre, Béatrice
- Journal of Structural Biology, Vol. 198, Issue 3
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
Be more specific! Laser-assisted microdissection of plant cells
journal, August 2005
- Day, Robert C.; Grossniklaus, Ueli; Macknight, Richard C.
- Trends in Plant Science, Vol. 10, Issue 8
Pathway Tools version 19.0 update: software for pathway/genome informatics and systems biology
journal, October 2015
- Karp, Peter D.; Latendresse, Mario; Paley, Suzanne M.
- Briefings in Bioinformatics, Vol. 17, Issue 5
Laser Capture Microdissection
journal, November 1996
- Emmert-Buck, Michael R.; Bonner, Robert F.; Smith, Paul D.
- Science, Vol. 274, Issue 5289
Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications
journal, April 2003
- Ahram, Mamoun; Flaig, Michael J.; Gillespie, John W.
- PROTEOMICS, Vol. 3, Issue 4
Single-Cell Genomic Analysis in Plants
journal, January 2018
- Yuan, Yuxuan; Lee, HueyTyng; Hu, Haifei
- Genes, Vol. 9, Issue 1
MS-GF+ makes progress towards a universal database search tool for proteomics
journal, October 2014
- Kim, Sangtae; Pevzner, Pavel A.
- Nature Communications, Vol. 5, Issue 1
Alcohol based fixatives provide excellent tissue morphology, protein immunoreactivity and RNA integrity in paraffin embedded tissue specimens
journal, April 2013
- Milcheva, Rositsa; Janega, Pavol; Celec, Peter
- Acta Histochemica, Vol. 115, Issue 3
Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples
journal, April 2018
- Zhu, Ying; Zhao, Rui; Piehowski, Paul D.
- International Journal of Mass Spectrometry, Vol. 427
Fundamentals of rapid injection molding for microfluidic cell-based assays
journal, January 2018
- Lee, Ulri N.; Su, Xiaojing; Guckenberger, David J.
- Lab on a Chip, Vol. 18, Issue 3
Towards Building a Plant Cell Atlas
journal, April 2019
- Rhee, Seung Y.; Birnbaum, Kenneth D.; Ehrhardt, David W.
- Trends in Plant Science, Vol. 24, Issue 4