Quantitative assay of targeted proteome in tomato trichome glandular cells using a large-scale selected reaction monitoring strategy

Ayako Takemori,Hiroshi Nonami,Hisashi Ômura,Taiken Nakashima,Yuki Tanaka,Nobuaki Takemori,Keisuke Nakata

doi:10.1186/s13007-019-0427-7

Ayako Takemori, Hiroshi Nonami + Show 5 more

Open Access

https://doi.org/10.1186/s13007-019-0427-7

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Abstract

BackgroundGlandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells. To systematically understand the metabolic processes in glandular cells, it is indispensable to analyze cellular proteome dynamics. The conventional proteomics methods based on mass spectrometry have enabled large-scale protein analysis, but require a large number of trichome samples for in-depth analysis and are not suitable for rapid and sensitive quantification of targeted proteins.ResultsHere, we present a high-throughput strategy for quantifying targeted proteins in specific trichome glandular cells, using selected reaction monitoring (SRM) assays. The SRM assay platform, targeting proteins in type VI trichome gland cells of tomato as a model system, demonstrated its effectiveness in quantifying multiple proteins from a limited amount of sample. The large-scale SRM assay uses a triple quadrupole mass spectrometer connected online to a nanoflow liquid chromatograph, which accurately measured the expression levels of 221 targeted proteins contained in the glandular cell sample recovered from 100 glandular trichomes within 120 min. Comparative quantitative proteomics using SRM assays of type VI trichome gland cells between different organs (leaves, green fruits, and calyx) revealed specific organ-enriched proteins.ConclusionsWe present a targeted proteomics approach using the established SRM assays which enables quantification of proteins of interest with minimum sampling effort. The remarkable success of the SRM assay and its simple experimental workflow will increase proteomics research in glandular trichomes.

Highlights

Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells
In the selected reaction monitoring (SRM) assay, the biological samples are fragmented by digestion with a site-specific protease, trypsin or Lys-C, and the derived peptides are used in the SRM assay
The mixture of digested peptides is primarily separated by liquid chromatography (LC), followed by high selective detection of the peptides derived from the target proteins by quadrupole mass spectrometry (MS) filters based on their mass to charge ratio

Summary

Introduction

Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells. The conventional proteomics methods based on mass spectrometry have enabled large-scale protein analysis, but require a large number of trichome samples for in-depth analysis and are not suitable for rapid and sensitive quantification of targeted proteins. Glandular trichomes are called natural cell factories because of their production of special metabolites, and they have high commercial value as food and pharmaceutical products [13,14,15]. For in-depth proteome analysis, it is necessary to recover a large amount of TGCs, which makes the analysis difficult when the available sample is limited

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