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Quantitative Assay of Targeted Proteome in Tomato Trichome Takemori et al. Plant Methods (2019) 15:40 https://doi.org/10.1186/s13007-019-0427-7 Plant Methods METHODOLOGY Open Access Quantitative assay of targeted proteome in tomato trichome glandular cells using a large-scale selected reaction monitoring strategy Ayako Takemori1, Taiken Nakashima2, Hisashi Ômura3, Yuki Tanaka4, Keisuke Nakata1, Hiroshi Nonami1,5,6 and Nobuaki Takemori4,6* Abstract Background: Glandular 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 quantifcation of targeted proteins. Results: Here, we present a high-throughput strategy for quantifying targeted proteins in specifc 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 efectiveness in quantifying multiple proteins from a lim- ited amount of sample. The large-scale SRM assay uses a triple quadrupole mass spectrometer connected online to a nanofow liquid chromatograph, which accurately measured the expression levels of 221 targeted proteins con- tained 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 diferent organs (leaves, green fruits, and calyx) revealed specifc organ-enriched proteins. Conclusions: We present a targeted proteomics approach using the established SRM assays which enables quantif- cation of proteins of interest with minimum sampling efort. The remarkable success of the SRM assay and its simple experimental workfow will increase proteomics research in glandular trichomes. Keywords: Tomato type VI glandular trichome, Secondary metabolism, Plant defense mechanism, Targeted proteomics, Mass spectrometry, Selected reaction monitoring, Proteotypic peptide, Protein quantifcation, AQUA peptide, QconCAT Background trichomes [2, 3]. Glandular trichomes are commonly Approximately 30% of vascular plants have epidermal composed of the base, stalk, and head units, and are protrusions called trichomes on their aerial parts [1]. further classifed into several subtypes based on their Multicellular trichomes that produce and store various morphology. Te glandular trichomes have one or more secondary metabolites are classifed as glandular-type secretory cells, called trichome glandular cells (TGCs), on the head, and various types of secondary metabo- lites including terpenoids, favonoids, and alkaloids are *Correspondence: [email protected] 4 Advanced Research Support Center, Ehime University, Toon 791-0295, synthesized in TGCs [4–9]. TGCs rapidly secrete intra- Japan cellular components in response to external physical Full list of author information is available at the end of the article stimulation, and the secreted components play important © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Takemori et al. Plant Methods (2019) 15:40 Page 2 of 13 roles, such as plant defense against insects and pathogens proteomics [35, 36], and its efectiveness has been dem- [10], protection against desiccation [11], and as a barrier onstrated in various model organisms [37–40]. Here, we against atmospheric oxidative stress [12]. Glandular tri- established an experimental workfow to develop a large- chomes are called natural cell factories because of their scale SRM assay for monitoring the TGC proteome and production of special metabolites, and they have high implemented targeted proteomics studies. We selected commercial value as food and pharmaceutical products cultivated tomato Solanum lycopersicum, for which high- [13–15]. quality genome sequence information is available [41], Several molecular omics studies have been conducted and analyzed TGCs for type VI trichomes, which is the to better understand metabolite production processes in most abundant type in tomato [3]. By using an estab- TGCs [16–20]. In particular, mass spectrometry (MS)- lished workfow, comprehensive quantitative information based analytical techniques have been used for highly on the targeted proteome can be easily acquired using a sensitive and high-throughput detection of cellular mol- small amount of TGC sample. In the present study, we ecules in TGCs. MS systems combined with separation assessed whether established SRM assays could be used techniques, e.g., gas chromatography [20, 21] or liquid for comparative proteomic analysis of TGCs in three dif- chromatography (LC) [22], are used extensively to gain ferent tomato organs. Further, we evaluated whether a insights into the metabolomic profles of various TGCs. combination of established assays and internal standards Besides the analysis of cellular metabolomes, the efec- could be used to quantify the absolute amounts of multi- tiveness of MS has also been demonstrated in large-scale ple target proteins in TGCs. analyses of proteins related to metabolite production in TGCs [23–28]. Current proteomics research is mostly Results based on an experimental approach using an LC-tandem Experimental strategies for SRM assay development MS (MS/MS), called shotgun proteomics [29]. Shotgun Figure 1 shows the workfow of the SRM assay developed proteomics is performed by sequencing the peptides to target TGC proteins. In the SRM assay, the biological derived from the protease digestion of proteins in bio- samples are fragmented by digestion with a site-specifc logical samples, enabling large-scale identifcation of pro- protease, trypsin or Lys-C, and the derived peptides are tein components. However, the complexity of the cellular used in the SRM assay. Te mixture of digested peptides proteome is high, and it is generally difcult to detect is primarily separated by LC, followed by high selective trace components reproducibly using shotgun proteom- detection of the peptides derived from the target proteins ics. Te amount of sample used for single shotgun anal- by quadrupole MS flters based on their mass to charge ysis depends on the capacity of the LC–MS system, but ratio. Terefore, the selection of target peptides with high a minimum of 10 μg of total protein as starting material ionization efciency and reproducible detection, called is generally required to identify more than 100 proteins. “proteotypic peptides” [42], is indispensable for develop- Terefore, for in-depth proteome analysis, it is neces- ing reliable assays. Due to the difculty of theoretical pre- sary to recover a large amount of TGCs, which makes the diction, it is desirable to select proteotypic peptides based analysis difcult when the available sample is limited. on the datasets from shotgun proteomics analysis. In this In addition to the shotgun approach, which aims to study, we frst conducted a shotgun proteomics analysis analyze as many proteins as possible, an alternative of TGCs by a gel electrophoresis-assisted LC–MS/MS approach to quantify only the targeted proteins with (GeLC–MS/MS) approach [43] and surveyed the peptide minimum steps in a sensitive manner is required in candidates suitable for high-throughput SRM. Based on TGC proteomics research. In this study, we focused on the obtained peptide information, we next established a a novel MS-based proteomics strategy using selected large-scale SRM assay targeting TGC proteins with pro- reaction monitoring (SRM), also known as multiple reac- teotypic peptides. tion monitoring (MRM), to obtain accurate quantitative information of target proteins from a small amount of GeLC–MS/MS analysis of TGC proteomes TGC sample. SRM has high sensitivity in terms of selec- Shotgun proteomics analysis was performed using glan- tively quantifying target molecules in complex analytical dular cells from type VI trichomes of tomato collected samples [30]. Currently, for protein quantifcation, the from diferent organs (the fruits and leaves). TGCs were LC-SRM assay using triple quadrupole MS combined collected manually under a stereo microscope using a with LC is used; with this approach multiple target pro- pair of forceps (Fig. 2a). TGCs were stably placed at the teins can be simultaneously detected with a single LC tip of the forceps, enabling easy recovery of cellular com- run [31–34]. Quantitative analysis using a large-scale ponents by immersing the tip rapidly in sodium dodecyl SRM assay covering a group of cellular proteins involved sulfate (SDS) lysis bufer. Te amount of protein con- in a specifc biological process is now called “targeted” tained in glandular cells/trichomes was diferent among Takemori et al. Plant Methods (2019) 15:40 Page 3 of 13 Fig. 1 Schematic overview
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