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

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 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.