Spatially Resolved Proteome Profiling of <200 Cells from Tomato Fruit Pericarp by Integrating Laser-Capture Microdissection with Nanodroplet Sample Preparation

Journal Article · Fri Aug 17 00:00:00 EDT 2018 · Analytical Chemistry

DOI:https://doi.org/10.1021/acs.analchem.8b03005· OSTI ID:1503564

Liang, Yiran ^[1]; Zhu, Ying  ^[1]; Dou, Maowei ^[1]; Xu, Kerui ^[1]; Chu, Rosalie K. ^[1]; Chrisler, William B. ^[2]; Zhao, Rui ^[1]; Hixson, Kim K. ^[1]; Kelly, Ryan T.  ^[3]

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1. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
2. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
3. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, United States; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States

Due to sensitivity limitations, global proteome measurements generally require large amounts of biological starting material, which masks heterogeneity within the samples and differential protein expression among constituent cell types. Methods for spatially-resolved proteomics are being developed to resolve protein expression for distinct single cell types among highly heterogeneous tissues, but have primarily been applied to mammalian systems. Here we evaluate the performance of cell-type-specific proteome analysis of tomato fruit pericarp tissues by a platform integrating laser capture microdissection (LCM) and a recently developed automated sample preparation system (nanoPOTS, Nanodroplet processing in One pot for Trace Samples). Tomato fruits were cryosectioned prior to LCM, and tissues were dissected and captured directly into nanoPOTS chips for processing. Following processing, samples were analyzed by nanoLC-MS/MS. Approximately 1900 unique peptides and 422 proteins were identified on average from ~0.04 mm2 tissues comprising ~ 8-15 parenchyma cells. Spatially-resolved proteome analyses were performed using cells of outer epidermis, collenchyma, and parenchyma. Using =200 cells, a total of 1,870 protein groups were identified and the various tissues were easily resolved. The results provide spatial and tissue-specific insights into key enzymes and pathways involved in carbohydrate transport and source-sink relationships in tomato fruit. Of note, at the time of fruit ripening studied here, we identified differentially abundant proteins throughout the pericarp related to chlorophyll biogenesis, photosynthesis, and especially transport.

• Research Organization: Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1503564
• Report Number(s): PNNL-SA-134717
• Journal Information: Analytical Chemistry, Vol. 90, Issue 18; ISSN 0003-2700
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English