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Allele-specific gene responses are picked up in this workflow as gene loci displaying genotype-specific differential expression that often have single amino acid polymorphisms. If the resources are sufficient, a genotype-specific mRNAseq database is recommended where a link is made to the allele-specific transcription levels. If the resources are limited, allele-specific proteins can be detected by the detection of genotype-specific peptides and the identification against existing genomics libraries of the parents.Tomato is a major crop plant and an important constituent of the human diet. Exclusive features such as bearing fleshy fruits and undergoing a phase transition from partially photosynthetic to fully heterotrophic metabolism make tomato fruit a model system for fruit development studies. Although the tomato genome has been completely sequenced, functional proteomics studies are still at their starting stage. Proteomics technologies, especially the combination of multiple approaches, provide a very powerful tool to accurately identify functional proteins and investigate certain sets of proteins in more detail. The direct binding of plant 14-3-3 proteins to their multiple target proteins modulates the functions of the latter, suggesting that these 14-3-3 proteins are directly involved in various physiological pathways. This chapter outline methods for the identification of 14-3-3 protein complexes in tomato fruit tissues. These methods include detailed protocols for protein extraction, coimmunoprecipitation, SDS-PAGE, SYPRO Ruby staining, in-gel trypsin digestion, and LC-MS/MS analysis for 14-3-3 interactomics.Cross-linking converts noncovalent interactions between proteins into covalent bonds. The now artificially fused molecules are stable during purification steps (e.g., immunoprecipitation). In combination with a variety of techniques, including Western blotting, mass spectrometry (MS), and bioinformatics, this technology provides improved opportunities for modelling structural details of functional complexes in living cells and protein-protein interaction networks. The presented strategy of immunoaffinity purification and mass spectrometry (AP-MS) coupled with in vivo cross-linking can easily be adapted as a robust workflow in interactome analyses of various species, also nonmodel organisms.Protein functions often rely on protein-protein interactions. Hence, knowledge about the protein interaction network is essential for an understanding of protein functions and plant physiology. A major challenge of the postgenomic era is the mapping of protein-protein interaction networks. This chapter describes a mass spectrometry-based label-free quantification approach to identify in vivo protein interaction networks. The procedure starts with the extraction of intact protein complexes from transgenic plants expressing the protein of interest fused to a GFP-Tag (bait-GFP), as well as plants expressing a free GFP as background control. Enrichment of the GFP-tagged protein together with its interaction partners, as well as the free GFP, is performed by immunoaffinity purification. The pull-down quality can be evaluated by simple gel-based techniques. In parallel, the captured proteins are trypsin-digested and relatively quantified by label-free mass spectrometry-based quantification. The relative quantification approach largely relies on the normalization of protein abundances of background-binding proteins, which occur in both bait-GFP and free GFP pull-downs. Therefore, relative quantification of the protein pull-down is superior over methods that solely rely on protein identifications and removal of often copurified high-abundance proteins from the bait-GFP pull-downs, which might remove real interaction partners. A further strength of this method is that it can be applied to any soluble GFP-tagged protein.Acetylation of lysine side chains at their ε-amino group is a reversible posttranslational modification (PTM), which can affect diverse protein functions. Lysine acetylation was first described on histones, and nowadays gains more and more attention due to its more general occurrence in proteomes, and its possible crosstalk with other protein modifications. Here we describe a workflow to investigate the acetylation of lysine-containing peptides on a large scale. For this high-resolution lysine acetylome analysis, dimethyl-labeled peptide samples are pooled and offline-fractionated using hydrophilic interaction liquid chromatography (HILIC). The offline fractionation is followed by an immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for data acquisition and subsequent data analysis.N-linked glycans are a ubiquitous posttranslational modification and are essential for correct protein folding in the endoplasmic reticulum of plants. However, this likely represents a narrow functional role for the diverse array of glycan structures currently associated with N-glycoproteins in plants. The identification of N-linked glycosylation sites and their structural characterization by mass spectrometry remains challenging due to their size, relative abundance, structural heterogeneity, and polarity. Current proteomic workflows are not optimized for the enrichment, identification and characterization of N-glycopeptides. Here we describe a detailed analytical procedure employing hydrophilic interaction chromatography enrichment, high-resolution tandem mass spectrometry employing complementary fragmentation techniques (higher-energy collisional dissociation and electron-transfer dissociation) and a data analytics workflow to produce an unbiased high confidence N-glycopeptide profile from plant samples.Parallel reaction monitoring (PRM) is a liquid chromatography-mass spectrometry (LC-MS)-based targeted peptide/protein quantification method that was initially implemented for Orbitrap mass spectrometers. Here, we describe detailed workflows that utilize the freely available MaxQuant and Skyline software packages to target peptides of interest, primarily focusing on phosphopeptides.The unicellular alga Chlamydomonas reinhardtii is a model photosynthetic organism for the study of microalgal processes. Along with genomic and transcriptomic studies, proteomic analysis of Chlamydomonas has led to an increased understanding of its metabolic signaling as well as a growing interest in the elucidation of its phosphorylation networks. To this end, mass spectrometry-based proteomics has made great strides in large-scale protein quantitation as well as analysis of posttranslational modifications (PTMs) in a high-throughput manner. An accurate quantification of dynamic PTMs, such as phosphorylation, requires high reproducibility and sensitivity due to the substoichiometric levels of modified peptides, which can make depth of coverage challenging. Here we present a method using TiO2-based phosphopeptide enrichment paired with label-free LC-MS/MS for phosphoproteome quantification. Three technical replicate samples in Chlamydomonas were processed and analyzed using this approach, quantifying a total of 1775 phosphoproteins with a total of 3595 phosphosites. With a median CV of 21% across quantified phosphopeptides, implementation of this method for differential studies provides highly reproducible analysis of phosphorylation events. While the culturing and extraction methods used are specific to facilitate coverage in algal species, this approach is widely applicable and can easily extend beyond algae to other photosynthetic organisms with minor modifications.Phosphorylation is a posttranslational reversible modification related to signaling and regulatory mechanisms. Protein phosphorylation is linked to structural changes that modulate protein activity, interaction, or localization and therefore the cell signaling pathways. The use of techniques for phosphoprotein enrichment along with mass spectrometry has become a powerful tool for the characterization of signal transduction in model organisms. However, limited efforts have focused on the establishment of protocols for the analysis of the phosphoproteome in nonmodel organisms such as tropical fruits. This chapter describes a potential pipeline for sample preparation and enrichment of phosphorylated proteins/peptides before MS analysis of peels of some species of tropical fruits.Most quantitative proteomics experiments either target a limited number of selected proteins for quantification or quantify proteins on a broad scale in an untargeted manner. However, we recently demonstrated that experiments that have both targeted and untargeted components can be particularly advantageous. Using a combined targeted and untargeted liquid chromatography-tandem mass spectrometry data acquisition strategy termed TDA/DDA (shorthand for targeted data acquisition/data-dependent acquisition), which we applied to a model quantitative plant proteomics experiment performed on Arabidopsis, we demonstrated improved quantification of both targeted and untargeted proteins relative to purely untargeted experiments performed using conventional data-dependent acquisition (Hart-Smith et al. Front Plant Sci 81669, 2017). This suggests that many quantitative proteomics datasets earmarked for collection using data-dependent acquisition are likely to benefit from the use of TDA/DDA instead.This chapter describes ed using data-independent acquisition.The proteomics of orphan, unsequenced, and recalcitrant organisms is highly challenging. This is the case of the typical Mediterranean forest tree Holm oak (Quercus ilex). Proteomics has moved on quite fast from the classical 2DE-MS to shotgun or gel-free/label-free approaches, with the latter possessing a series of advantages over the gel-based ones. Before translating proteomics data into biological knowledge, a few questions as to the analysis technique itself have to be answered including its confidence in protein identification and quantification. It is important to clearly differentiate a hit from an ortholog and gene product identification, with the difference depending on the database and the confidence parameters (score, number of peptides, and coverage). selleck products With respect to quantification and for comparative purposes it is important to make sure that we are within the linear dynamic range. For that, a calibration curve based on mass spectrometry analysis of a serial dilution of the extracts should be performed. Thus, just by validating our data with the aim of improving the quality of the analysis enables us to give a correct interpretation of our results. We show a method that aims to improve the confidence in protein identification and quantification in the orphan species Q. ilex using a shotgun proteomics approach.Proteins produce or regulate nearly every component of cells. Thus, the ability to quantitatively determine the protein abundance and posttranslational modification (PTM) state is a critical aspect toward our understanding of biological processes. In this chapter, we describe methods to globally quantify protein abundance and phosphorylation state using isobaric labeling with tandem mass tags followed by phosphopeptide enrichment.Dimethyl labeling is a type of stable-isotope labeling suitable for creating isotopic variants of peptides and thus be utilized for quantitative proteomics experiments. Labeling is achieved through a reductive amination/alkylation reaction using the low-cost reagents formaldehyde and cyanoborohydride, resulting in dimethylation of free amine groups of Lys and N-termini. Availability of isotopomeric forms of these reagents allows for the generation of up to six different isotopic variants. Here we describe the application of dimethylation to create two isotopic variants, light and heavy, differing in 4 Da, to label the total tryptic digest peptides of cocoa pod extracted from healthy pods from cultivars susceptible and resistant to the fungal disease called "frosty pod" caused by Moniliophthora roreri.
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