Quantitative assay of targeted proteome in trichome glandular cells using large-scale selected reaction monitoring strategy
Takemori A, Nakashima T, Ômura H, Tanaka Y, Nakata K, Nonami H, Takemori N. Quantitative assay of targeted proteome in tomato trichome glandular cells using a large-scale selected reaction monitoring strategy. Plant Methods. 2019 Apr 24;15:40. doi: 10.1186/s13007-019-0427-7
- Organism: Solanum lycopersicum
- Instrument: QTRAP 5500
Tomato type VI trichome glandular cell
Lab head: Nobuaki Takemori
Submitter: Nobuaki Takemori
Background: Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in the glandular cell. In order to systematically understand the metabolic processes in the glandular cells, it is indispensable to analyze the cellular proteome dynamics. The conventional proteomics methods based on mass spectrometry have been enabled large-scale protein analysis, but requires a large number of trichome samples for in-depth analysis and is not suitable for rapid and sensitive quantification of targeted proteins.
Results: Here we present a high-throughput strategy for quantifying targeted proteins in a specific trichome glandular cell, using selected reaction monitoring (SRM) assay. SRM assay platform, targeting the proteins in tomato type VI trichome gland cells as a model study, demonstrated its effectiveness in multiple protein quantitation from a limited sample amount. A large-scale SRM assay developed using triple quadrupole mass spectrometer online connected to nanoflow liquid chromatography has enough capability to accurately measure the expression level of 221 targeted proteins contained in the glandular cell sample recovered from the 100 glandular trichomes within 120 minutes.
Conclusions: Targeted proteomics approach using established SRM assays makes it possible to quantify the proteins of interest with minimum effort in the sampling. The remarkable success of the SRM assay developments by a simple experimental workflow will increase the plant biologist's accessibility to proteomics research for glandular trichome.
The development of SRM assays was performedwith the Skyline software. The database search files from the ProteinPilot software (SCIEX) were used to build the MS spectral library containing only tryptic digested peptides (allowed one missed cleavage) with their confidence value > 0.9. The analysis of obtained SRM data was performed with Skyline. Endogenous peptide peaks in the acquired SRM chromatogram were selected manually based on chromatographic elution pattern and the dot product (dotp) value.
Tomato cultivar ‘Micro-Tom’ (S. lycopersicum L. cv. Microtom) was used for all the studies.
Created on 1/7/19, 9:05 PM