Cell‐Type‐Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform

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.

In-Depth Mass Spectrometry-Based Proteomics of Formalin-Fixed, Paraffin-Embedded Tissues with a Spatial Resolution of 50–200 μm

“…These general conditions have been used extensively for frozen tissues. [17][18][19][20]22,23 As shown in Figure 2 and the first two rows of Table 1 (frozen and FFPE 1), these extraction conditions yielded reductions of 51 and 35% in peptide and proteome coverage (excluding match between run values), respectively, for the FFPE samples relative to frozen tissues, indicating poor suitability of these conditions for FFPE.…”

“…Using the single-pot, solid-phase-enhanced (SP3) sample processing method, approximately 1500 proteins were identified from 10 μm thick brain tissue sections with a 0.06 mm 2 area . Our group has also focused on improving the sensitivity of MS-based proteomics for low-input samples including single cells − and trace tissue samples − through improvements in sample preparation, separations, ionization, and mass spectrometry. To minimize losses during sample preparation, we developed nanodroplet processing in one pot for trace samples (nanoPOTS), which miniaturizes processing volumes to the nanoliter scale to minimize surface exposure and transfer losses while enhancing reaction kinetics.…”

Hanging drop sample preparation improves sensitivity of spatial proteomics

“…27 The platform has been widely used to study the cell-typespecific proteomes in rat and human brains, 26,28 mouse uterus, 27 tomato fruit, 29 and poplar leaf and root. 30 Herein, we describe an improved LCM-nanoPOTS spatial proteomics workflow. Specifically, we developed a hangingdrop (HD)-based sample preparation approach by simply positioning the nanoPOTS chip upside-down during protein extraction and tryptic digestion steps.…”

“…27 The platform has been widely used to study the cell-type-specific proteomes in rat and human brains, 26,28 mouse uterus, 27 tomato fruit, 29 and poplar leaf and root. 30…”

Hanging-droplet sample preparation improves sensitivity of spatial proteomics

“…The platform has been widely used to study the cell-type-specific proteomes in rat and human brains, 26,27 mouse uterus, 28 tomato fruit, 29 and poplar leaf and root. 30 Herein, we describe an improved LCM-nanoPOTS spatial proteomics workflow. Specifically, we developed a hangingdrop (HD)-based sample preparation approach by simply positioning the nanoPOTS chip upside-down during protein extraction and tryptic digestion steps.…”