Cell-Type-Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform
Vimal K. Balasubramanian
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Contribution: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - original draft
Search for more papers by this authorSamuel O. Purvine
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Contribution: Data curation, Formal analysis, Investigation, Methodology, Software, Writing - original draft, Writing - original draft
Search for more papers by this authorYiran Liang
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah
Contribution: Investigation, Methodology, Writing - original draft, Writing - original draft
Search for more papers by this authorRyan T. Kelly
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah
Search for more papers by this authorLjiljana Pasa-Tolic
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Contribution: Resources, Supervision, Writing - original draft
Search for more papers by this authorWilliam B. Chrisler
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Contribution: Investigation, Methodology, Writing - original draft
Search for more papers by this authorEduardo Blumwald
Department of Plant Sciences, University of California, Davis, California
Contribution: Funding acquisition, Investigation, Project administration, Writing - original draft
Search for more papers by this authorC. Neal Stewart Jr.
Department of Plant Sciences, Center for Agricultural Synthetic Biology, University of Tennessee, Knoxville, Tennessee
Contribution: Funding acquisition, Project administration, Writing - original draft
Search for more papers by this authorCorresponding Author
Ying Zhu
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Corresponding author: [email protected]; [email protected]
Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Writing - original draft, Writing - original draft
Search for more papers by this authorCorresponding Author
Amir H. Ahkami
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Corresponding author: [email protected]; [email protected]
Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing - original draft, Writing - original draft
Search for more papers by this authorVimal K. Balasubramanian
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Contribution: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - original draft
Search for more papers by this authorSamuel O. Purvine
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Contribution: Data curation, Formal analysis, Investigation, Methodology, Software, Writing - original draft, Writing - original draft
Search for more papers by this authorYiran Liang
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah
Contribution: Investigation, Methodology, Writing - original draft, Writing - original draft
Search for more papers by this authorRyan T. Kelly
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah
Search for more papers by this authorLjiljana Pasa-Tolic
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Contribution: Resources, Supervision, Writing - original draft
Search for more papers by this authorWilliam B. Chrisler
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Contribution: Investigation, Methodology, Writing - original draft
Search for more papers by this authorEduardo Blumwald
Department of Plant Sciences, University of California, Davis, California
Contribution: Funding acquisition, Investigation, Project administration, Writing - original draft
Search for more papers by this authorC. Neal Stewart Jr.
Department of Plant Sciences, Center for Agricultural Synthetic Biology, University of Tennessee, Knoxville, Tennessee
Contribution: Funding acquisition, Project administration, Writing - original draft
Search for more papers by this authorCorresponding Author
Ying Zhu
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Corresponding author: [email protected]; [email protected]
Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Writing - original draft, Writing - original draft
Search for more papers by this authorCorresponding Author
Amir H. Ahkami
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington
Corresponding author: [email protected]; [email protected]
Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing - original draft, Writing - original draft
Search for more papers by this authorAbstract
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
Open Research
Data Availability Statement
Data sharing not applicable—no new data generated.
Supporting Information
| Filename | Description |
|---|---|
| cpz1153-sup-0001-TableS1.xlsx3.6 MB | Supplementary Information |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Literature Cited
- Ahram, M., Flaig, M. J., Gillespie, J. W., Duray, P. H., Linehan, W. M., Ornstein, D. K. … Emmert-Buck, M. R. (2003). Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications. Proteomics, 3, 413–421. doi: 10.1002/pmic.200390056.
- Baginsky, S. (2009). Plant proteomics: Concepts, applications, and novel strategies for data interpretation. Mass Spectrometry Reviews, 28, 93–120. doi: 10.1002/mas.20183.
- Birnbaum, K., Shasha, D. E., Wang, J. Y., Jung, J. W., Lambert, G. M., Galbraith, D. W., & Benfey P. N. (2003). A gene expression map of the Arabidopsis root. Science, 302, 1956–1960. doi: 10.1126/science.1090022.
- Böhmer, M., & Schroeder, J. I. (2011). Quantitative transcriptomic analysis of abscisic acid-induced and reactive oxygen species-dependent expression changes and proteomic profiling in Arabidopsis suspension cells. Plant Journal, 67, 105–118. doi: 10.1111/j.1365-313X.2011.04579.x.
- Chambers, M. C., Maclean, B., Burke, R., Amodei, D., Ruderman, D. L., Neumann, S. … Mallick, P. (2012). A cross-platform toolkit for mass spectrometry and proteomics. Nature Biotechnology, 30, 918–920. doi: 10.1038/nbt.2377.
- Chen, Q., Huang, R., Xu, Z., Zhang, Y., Li, L., Fu, J. … Gu, R. (2020). Label-free comparative proteomic analysis combined with laser-capture microdissection suggests important roles of stress responses in the black layer of maize kernels. International Journal of Molecular Sciences, 21(4), 1369. doi: 10.3390/ijms21041369.
- Collado-Romero, M., Alós, E., & Prieto, P. (2014). Unravelling the proteomic profile of rice meiocytes during early meiosis. Frontiers in Plant Science, 5, 356–356. doi: 10.3389/fpls.2014.00356.
- Cong, Y., Motamedchaboki, K., Misal, S. A., Liang, Y., Guise, A. J., Truong, T. … Kelly, R. T. (2021). Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell. Chemical Science, 12, 1001–1006. doi: 10.1039/D0SC03636F.
- Dai, S., & Chen, S. (2012). Single-cell-type proteomics: Toward a holistic understanding of plant function. Molecular and Cellular Proteomics, 11, 1622–1630. doi: 10.1074/mcp.R112.021550.
- Dai, S., Li, L., Chen, T., Chong, K., Xue, Y., & Wang, T. (2006). Proteomic analyses of Oryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth. Proteomics, 6, 2504–2529. doi: 10.1002/pmic.200401351.
- Dannhorn, A., Kazanc, E., Ling, S., Nikula, C., Karali, E., Serra, M. P. … Takats, Z. (2020). Universal sample preparation unlocking multimodal molecular tissue imaging. Analytical Chemistry, 92, 11080–11088. doi: 10.1021/acs.analchem.0c00826.
- Day, R. C., Grossniklaus, U., & Macknight, R. C. (2005). Be more specific! Laser-assisted microdissection of plant cells. Trends in Plant Science, 10, 397–406. doi: 10.1016/j.tplants.2005.06.006.
- Dembinsky, D., Woll, K., Saleem, M., Liu, Y., Fu, Y., Borsuk, L. A. … Hochholdinger, F. (2007). Transcriptomic and proteomic analyses of pericycle cells of the maize primary root. Plant Physiology, 145, 575–588. doi: 10.1104/pp.107.106203.
- Dou, M., Tsai, C.-F., Piehowski, P. D., Wang, Y., Fillmore, T. L., Zhao, R. … Zhu, Y. (2019). Automated nanoflow two-dimensional reversed-phase liquid chromatography system enables in-depth proteome and phosphoproteome profiling of nanoscale samples. Analytical Chemistry, 91, 9707–9715. doi: 10.1021/acs.analchem.9b01248.
- Elias, J. E., & Gygi, S. P. (2010). Target-decoy search strategy for mass spectrometry-based proteomics. Methods in Molecular Biology, 604, 55–71. doi: 10.1007/978-1-60761-444-9_5.
- Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., Chuaqui, R. F., Zhuang, Z., Goldstein, S. R. … Liotta L. A. (1996). Laser capture microdissection. Science, 274, 998–1001. doi: 10.1126/science.274.5289.998.
- Evers, D. L., He, J., Kim, Y. H., Mason, J. T., & O'Leary, T. J. (2011). Paraffin embedding contributes to RNA aggregation, reduced RNA yield, and low RNA quality. The Journal of Molecular Diagnostics, 13, 687–694. doi: 10.1016/j.jmoldx.2011.06.007.
- Hashiguchi, A., & Komatsu, S. (2016). Impact of post-translational modifications of crop proteins under abiotic stress. Proteomes, 4, 42. doi: 10.3390/proteomes4040042.
- Hölscher, D., & Schneider, B. (2008). Application of laser-assisted microdissection for tissue and cell-specific analysis of RNA, proteins, and metabolites. In U. Lüttge, W. Beyschlag, & J. Murata (Eds.), Progress in Botany (pp. 141–167). Berlin, Heidelberg: Springer Berlin Heidelberg.
- Hu, J., Rampitsch, C., & Bykova, N. V. (2015). Advances in plant proteomics toward improvement of crop productivity and stress resistance. Frontiers in Plant Science, 6, 209–209. doi: 10.3389/fpls.2015.00209.
- Ishimaru, T., Nakazono, M., Masumura, T., Abiko, M., San-oh, Y., Nishizawa, N. K., & Kondo, M. (2007). A method for obtaining high integrity RNA from developing aleurone cells and starchy endosperm in rice (Oryza sativa L.) by laser microdissection. Plant Science, 173, 321–326. doi: 10.1016/j.plantsci.2007.06.004.
- Karp, P. D., Latendresse, M., Paley, S. M., Krummenacker, M., Ong, Q. D., Billington, R. … Caspi R. (2016). Pathway Tools version 19.0 update: Software for pathway/genome informatics and systems biology. Briefings in Bioinformatics, 17, 877–890. doi: 10.1093/bib/bbv079.
- Kersten, B., Faivre Rampant, P., Mader, M., Le Paslier, M. C., Bounon, R., Berard, A. … Fladung, M. (2016). Genome sequences of Populus tremula chloroplast and mitochondrion: Implications for holistic poplar breeding. PloS One, 11, e0147209. doi: 10.1371/journal.pone.0147209.
- Kim, S., & Pevzner, P. A. (2014). MS-GF+ makes progress towards a universal database search tool for proteomics. Nature Communication, 5, 5277. doi: 10.1038/ncomms6277.
- Komatsu, S., & Hossain, Z. (2013). Organ-specific proteome analysis for identification of abiotic stress response mechanism in crop. Frontiers in Plant Science, 4, 71. doi: 10.3389/fpls.2013.00071.
- Krasensky, J., & Jonak, C. (2012). Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. Journal of Experimental Botany, 63, 1593–1608. doi: 10.1093/jxb/err460.
- Lee, U. N., Su, X., Guckenberger, D. J., Dostie, A. M., Zhang, T., Berthier, E., & Theberge, A. B. (2018). Fundamentals of rapid injection molding for microfluidic cell-based assays. Lab on a Chip, 18, 496–504. doi: 10.1039/C7LC01052D.
- Lei, Z., Elmer, A. M., Watson, B. S., Dixon, R. A., Mendes, P. J., & Sumner, L. W. (2005). A two-dimensional electrophoresis proteomic reference map and systematic identification of 1367 proteins from a cell suspension culture of the model legume Medicago truncatula. Molecular and Cellular Proteomics, 4, 1812–1825. doi: 10.1074/mcp.D500005-MCP200.
- Liang, Y., Zhu, Y., Dou, M., Xu, K., Chu, R. K., Chrisler, W. B. … Kelly, R. T. (2018). Spatially resolved proteome profiling of <200 cells from tomato fruit pericarp by integrating laser-capture microdissection with nanodroplet sample preparation. Analytical Chemistry, 90, 11106–11114. doi: 10.1021/acs.analchem.8b03005.
- Libault, M., Pingault, L., Zogli, P., & Schiefelbein, J. (2017). Plant systems biology at the single-cell level. Trends in Plant Science, 22, 949–960. doi: 10.1016/j.tplants.2017.08.006.
- Liu, W. W., Zhu, Y., Feng, Y. M., Fang, J., & Fang, Q. (2017). Droplet-based multivolume digital polymerase chain reaction by a surface-assisted multifactor fluid segmentation approach. Analytical Chemistry, 89, 822–829. doi: 10.1021/acs.analchem.6b03687.
- Liu, Y., Lu, S., Liu, K., Wang, S., Huang, L., & Guo, L. (2019). Proteomics: A powerful tool to study plant responses to biotic stress. Plant Methods, 15, 135. doi: 10.1186/s13007-019-0515-8.
- Mader, M., Paslier, M.-C. Le, Bounon, R., Bérard, A., Rampant, P. F., Fladung, M. … Kersten, B. (2016). Whole-genome draft assembly of Populus tremula × P. alba clone INRA 717-1B4. Silvae Genetica, 65, 74–79. doi: 10.1515/sg-2016-0019.
- Marion, J., Bars, R. Le, Satiat-Jeunemaitre, B., & Boulogne, C. (2017). Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots. Journal of Structural Biology, 198, 196–202. doi: 10.1016/j.jsb.2017.03.008.
- Martin, L., Nicolas, P., Matas Arroyo, A., Shinozaki, Y., Catala, C., & Rose, J. (2016). Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling. Nature Protocols, 11, 2376–2388. doi: 10.1038/nprot.2016.146.
- Matas, A. J., Yeats, T. H., Buda, G. J., Zheng, Y., Chatterjee, S., Tohge, T. … Rose, J. K. C. (2011). Tissue- and cell-type specific transcriptome profiling of expanding tomato fruit provides insights into metabolic and regulatory specialization and cuticle formation. The Plant Cell, 23, 3893. doi: 10.1105/tpc.111.091173.
- Milcheva, R., Janega, P., Celec, P., Russev, R., & Babál, P. (2013). Alcohol based fixatives provide excellent tissue morphology, protein immunoreactivity and RNA integrity in paraffin embedded tissue specimens. Acta Histochemica, 115, 279–289. doi: 10.1016/j.acthis.2012.08.002.
- Monroe, M. E., Shaw, J. L., Daly, D. S., Adkins, J. N., & Smith, R. D. (2008). MASIC: A software program for fast quantitation and flexible visualization of chromatographic profiles from detected LC–MS(/MS) features. Computational Biology and Chemistry, 32, 215–217. doi: 10.1016/j.compbiolchem.2008.02.006.
- Nelson, T., Tausta, S. L., Gandotra, N., & Liu, T. (2006). Laser microdissection of plant tissue: What you see is what you get. Annual Review of Plant Biology, 57, 181–201. doi: 10.1146/annurev.arplant.56.032604.144138.
- Nestler, J., Schütz, W., & Hochholdinger, F. (2011). Conserved and unique features of the maize (Zea mays L.) root hair proteome. Journal of Proteome Research, 10, 2525–2537. doi: 10.1021/pr200003k.
- Petricka, J. J., Schauer, M. A., Megraw, M., Breakfield, N. W., Thompson, J. W., Georgiev, S. … Benfey, P. N. (2012). The protein expression landscape of the Arabidopsis root. Proceedings of the National Academy of Sciences, 109, 6811–6818. doi: 10.1073/pnas.1202546109.
- Polpitiya, A. D., Qian, W.-J., Jaitly, N., Petyuk, V. A., Adkins, J. N., Camp, D. G., II … Smith, R. D. (2008). DAnTE: A statistical tool for quantitative analysis of -omics data. Bioinformatics, 24, 1556–1558. doi: 10.1093/bioinformatics/btn217.
- Rhee, S. Y., Birnbaum, K. D., & Ehrhardt, D. W. (2019). Towards building a plant cell atlas. Trends in Plant Science, 24, 303–310. doi: 10.1016/j.tplants.2019.01.006.
- Ryu, K. H., Huang, L., Kang, H. M., & Schiefelbein, J. (2019). Single-cell RNA sequencing resolves molecular relationships among individual plant cells. Plant Physiology, 179, 1444–1456. doi: 10.1104/pp.18.01482.
- Schad, M., Lipton, M. S., Giavalisco, P., Smith, R. D., & Kehr, J. (2005). Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples. Electrophoresis, 26, 2729–2738. doi: 10.1002/elps.200410399.
- Schilmiller, A. L., Miner, D. P., Larson, M., McDowell, E., Gang, D. R., Wilkerson, C., & Last, R. L. (2010). Studies of a biochemical factory: Tomato trichome deep expressed sequence tag sequencing and proteomics. Plant Physiology, 153, 1212–1223. doi: 10.1104/pp.110.157214.
- Shaar-Moshe, L., Blumwald, E., & Peleg, Z. (2017). Unique physiological and transcriptional shifts under combinations of salinity, drought, and heat. Plant Physiology, 174, 421–434. doi: 10.1104/pp.17.00030.
- Shah, P., Zhang, B., Choi, C., Yang, S., Zhou, J., Harlan, R. … Zhang, H. (2015). Tissue proteomics using chemical immobilization and mass spectrometry. Analytical Biochemistry, 469, 27–33. doi: 10.1016/j.ab.2014.09.017.
- Shao, H.-B., Chu, L.-Y., Jaleel, C. A., & Zhao, C.-X. (2008). Water-deficit stress-induced anatomical changes in higher plants. Comptes Rendus Biologies, 331, 215–225. doi: 10.1016/j.crvi.2008.01.002.
- Sheoran, I. S., Ross, A. R., Olson, D. J., & Sawhney, V. K. (2007). Proteomic analysis of tomato (Lycopersicon esculentum) pollen. Journal of Experimental Botany, 58, 3525–3535. doi: 10.1093/jxb/erm199.
- Slavov, N. (2020). Unpicking the proteome in single cells. Science, 367, 512–513. doi: 10.1126/science.aaz6695.
- Solomon, M. J., & Varshavsky, A. (1985). Formaldehyde-mediated DNA-protein crosslinking: A probe for in vivo chromatin structures. Proceedings of the National Academy of Sciences of the United States of America, 82, 6470–6474. doi: 10.1073/pnas.82.19.6470.
- Sui, X., Nie, J., Li, X., Scanlon, M. J., Zhang, C., Zheng, Y. … Zhang, Z. (2018). Transcriptomic and functional analysis of cucumber (Cucumis sativus L.) fruit phloem during early development. The Plant Journal, 96, 982–996. doi: 10.1111/tpj.14084.
- Tang, W., & Tang, A. Y. (2019). Biological significance of RNA-seq and single-cell genomic research in woody plants. Journal of Forestry Research, 30, 1555–1568. doi: 10.1007/s11676-019-00933-w.
- Tsai, C. F., Zhao, R., Williams, S. M., Moore, R. J., Schultz, K., Chrisler, W. B. … Liu, T. (2020). An improved boosting to amplify signal with isobaric labeling (iBASIL) strategy for precise quantitative single-cell proteomics. Molecular & Cellular Proteomics, 19, 828–838. doi: 10.1074/mcp.RA119.001857.
- Tuskan, G. A., DiFazio, S., Jansson, S., Bohlmann, J., Grigoriev, I., Hellsten, U. … Rokhsar, D. (2006). The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science, 313, 1596–1604. doi: 10.1126/science.1128691.
- Van Cutsem, E., Simonart, G., Degand, H., Faber, A. M., Morsomme, P., & Boutry, M. (2011). Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response. Proteomics, 11, 440–454. doi: 10.1002/pmic.201000356.
- Wang, J., Yao, L., Li, B., Meng, Y., Ma, X., Lai, Y. … Wang, H. (2016). Comparative proteomic analysis of cultured suspension cells of the halophyte Halogeton glomeratus by iTRAQ provides insights into response mechanisms to salt stress. Frontiers in Plant Science, 7, 110–110. doi: 10.3389/fpls.2016.00110.
- Williams, S. M., Liyu, A. V., Tsai, C. F., Moore, R. J., Orton, D. J., Chrisler, W. B. … Zhu, Y. (2020). Automated coupling of nanodroplet sample preparation with liquid chromatography−mass spectrometry for high-throughput single-cell proteomics. Analytical Chemistry, 92, 10588–10596. doi: 10.1021/acs.analchem.0c01551.
- Yang, S., Li, H., Bhatti, S., Zhou, S., Yang, Y., Fish, T., & Thannhauser, T. W. (2020). The Al-induced proteomes of epidermal and outer cortical cells in root apex of cherry tomato ‘LA 2710’. Journal of Proteomics, 211, 103560. doi: 10.1016/j.jprot.2019.103560.
- Yi, L., Piehowski, P. D., Shi, T., Smith, R. D., & Qian, W. J. (2017). Advances in microscale separations towards nanoproteomics applications. Journal of Chromatography. A, 1523, 40–48. doi: 10.1016/j.chroma.2017.07.055.
- Yuan, Y., Lee, H., Hu, H., Scheben, A., & Edwards, D. (2018). Single-cell genomic analysis in plants. Genes (Basel), 9, 50. doi: 10.3390/genes9010050.
- Zhu, M., Dai, S., McClung, S., Yan, X., & Chen, S. (2009). Functional differentiation of Brassica napus guard cells and mesophyll cells revealed by comparative proteomics. Molecular & Cellular Proteomics, 8, 752–766. doi: 10.1074/mcp.M800343-MCP200.
- Zhu, Y., Clair, G., Chrisler, W. B., Shen, Y., Zhao, R., Shukla, A. K. … Kelly, R. T. (2018). Proteomic analysis of single mammalian cells enabled by microfluidic nanodroplet sample preparation and ultrasensitive NanoLC-MS. Angewandte Chemie, 57, 12370–12374. doi: 10.1002/anie.201802843.
- Zhu, Y., Dou, M., Piehowski, P. D., Liang, Y., Wang, F., Chu, R. K. … Kelly, R. T. (2018). Spatially resolved proteome mapping of laser capture microdissected tissue with automated sample transfer to nanodroplets. Molecular & Cellular Proteomics, 17, 1864–1874. doi: 10.1074/mcp.TIR118.000686.
- Zhu, Y., Li, H., Bhatti, S., Zhou, S., Yang, Y., Fish, T., & Thannhauser, T. W. (2016). Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots. Horticulture Research, 3, 16026. doi: 10.1038/hortres.2016.26.
- Zhu, Y., Piehowski, P. D., Zhao, R., Chen, J., Shen, Y., Moore, R. J. … Kelly, R. T. (2018). Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells. Nature Communications, 9, 882. doi: 10.1038/s41467-018-03367-w.
- Zhu, Y., Zhao, R., Piehowski, P. D., Moore, R. J., Lim, S., Orphan, V. J. … Kelly, R. T. (2018). Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples. International Journal of Mass Spectrometry, 427, 4–10. doi: 10.1016/j.ijms.2017.08.016.
- Zou, J., Song, L., Zhang, W., Wang, Y., Ruan, S., & Wu, W. H. (2009). Comparative proteomic analysis of Arabidopsis mature pollen and germinated pollen. Journal of Integrative Plant Biology, 51, 438–455. doi: 10.1111/j.1744-7909.2009.00823.x.
