iTRAQ-based quantitative analysis of protein mixtures with large fold change and dynamic range

Casado‐Vela, Juan; Martínez-Esteso, María José; Rodríguez, Eva Sancho; Borràs, Eva; Elortza, Félix; Bru-Martínez, Roque

doi:10.1002/pmic.200900509

Cited by 63 publications

(52 citation statements)

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“…Isobaric tags for relative and absolute protein quantitation (iTRAQ) are currently being used successfully to characterize and quantify changes in protein levels in complex biological samples [38]. The use of iTRAQ has become a consolidated technique in quantitative proteomics since large fold changes of protein expression within broad dynamic ranges of protein abundance can be measured quite accurately [39]. The iTRAQ method allows the multiplexed identification and quantitation of proteins in four different samples (4-plex) and has been recently scaled up to measure protein changes in up to eight different samples (8-plex) [40].…”

Section: Introductionmentioning

confidence: 99%

iTRAQ-based protein profiling provides insights into the central metabolism changes driving grape berry development and ripening

et al. 2013

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BackgroundGrapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components such as sugars, acids, flavors, anthocyanins, tannins, etc., accumulate in the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance in our understanding of berry development and ripening processes.ResultsWe report the developmental analysis of Vitis vinifera cv. Muscat Hamburg berries at the protein level from fruit set to full ripening. An iTRAQ-based bottom-up proteomic approach followed by tandem mass spectrometry led to the identification and quantitation of 411 and 630 proteins in the green and ripening phases, respectively. Two key points in development relating to changes in protein level were detected: end of the first growth period (7 mm-to-15 mm) and onset of ripening (15 mm-to-V100, V100-to-110). A functional analysis was performed using the Blast2GO software based on the enrichment of GO terms during berry growth.ConclusionsThe study of the proteome contributes to decipher the biological processes and metabolic pathways involved in the development and quality traits of fruit and its derived products. These findings lie mainly in metabolism and storage of sugars and malate, energy-related pathways such as respiration, photosynthesis and fermentation, and the synthesis of polyphenolics as major secondary metabolites in grape berry. In addition, some key steps in carbohydrate and malate metabolism have been identified in this study, i.e., PFP-PFK or SuSy-INV switches among others, which may influence the final sugar and acid balance in ripe fruit. In conclusion, some proteins not reported to date have been detected to be deregulated in specific tissues and developmental stages, leading to formulate new hypotheses on the metabolic processes underlying grape berry development. These results open up new lines to decipher the processes controlling grape berry development and ripening.

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Section: Introductionmentioning

confidence: 99%

iTRAQ-based protein profiling provides insights into the central metabolism changes driving grape berry development and ripening

et al. 2013

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“…To better appreciate the molecular consequences of this stress-induced SUMOylation, we developed a quantitative proteomic method amenable to intact plants that would help identify the most robustly regulated substrates (up or down). In order to avoid the inherent difficulties of SILAC MS (46, 47), we instead employed the iTRAQ labeling system to measure the relative abundance of specific peptides via tandem MS (48,49). This approach was combined with a threestep protocol to purify SUMO1 conjugates under stringent denaturing conditions (6 M guanidine-HCl and 8 M urea) from transgenic plants that were engineered to replace the two essential SUMO isoforms involved in stress-induced SUMOylation (SUMO1 and SUMO2 (8)) with a 6His-SUMO1-H89R variant designed for faithful genetic rescue, efficient purification, and better mapping of SUMO attachment sites (41).…”

Section: Development Of a Quantitative Proteomic Strategy To Monitor mentioning

confidence: 99%

“…Although SILAC-MS has advantages for cell cultures that permit facile protein labeling via the addition of isotopically tagged amino acids to the growth medium, its use remains challenging for intact organisms generally, and for plants in particular, which naturally synthesize the full complement of amino acids (46,47). To overcome these difficulties, we combined a stringent purification method for SUMO conjugates with the isobaric tag for relative and absolute quantification (iTRAQ) method (48,49) and an advanced SUMO-based normalization strategy to accurately measure the changes in SUMOylation state for individual substrates in intact Arabidopsis plants under stress. By tracking SUMO1/2 substrates before, during, and after a mild heat shock (37°C), or following oxidative and ethanol stress, we identified a catalogue of substrates that are robustly SUMOylated after environmental challenges.…”

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Quantitative Proteomics Reveals Factors Regulating RNA Biology as Dynamic Targets of Stress-induced SUMOylation in Arabidopsis

Miller

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Rytz

et al. 2013

Molecular & Cellular Proteomics

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The stress-induced attachment of small ubiquitin-like modifier (SUMO) to a diverse collection of nuclear proteins regulating chromatin architecture, transcription, and RNA biology has been implicated in protecting plants and animals against numerous environmental challenges. In order to better understand stress-induced SUMOylation, we combined stringent purification of SUMO conjugates with isobaric tag for relative and absolute quantification mass spectrometry and an advanced method to adjust for sample-to-sample variation so as to study quantitatively the SUMOylation dynamics of intact Arabidopsis seedlings subjected to stress. Inspection of 172 SUMO substrates during and after heat shock (37°C) revealed that stress mostly increases the abundance of existing conjugates, as opposed to modifying new targets. Some of the most robustly up-regulated targets participate in RNA processing and turnover and RNA-directed DNA modification, thus implicating SUMO as a regulator of the transcriptome during stress. Many of these targets were also strongly SUMOylated during ethanol and oxidative stress, suggesting that their modification is crucial for general stress tolerance. Collectively, our quantitative data emphasize the importance of SUMO to RNA-related processes protecting plants from adverse environments. The ability of cellular organisms to cope with environmental challenges requires the detection and immediate initiation of defense responses designed to mitigate the damage inflicted and enhance the organism's ability to tolerate future insults. For sessile organisms such as plants, robust stress responses are fundamental to their survival in a wide range of adverse environments (1, 2). Genetic and biochemical studies have identified a plethora of input pathways and output responses for stress protection in both prokaryotes and eukaryotes. Universally important is the synthesis of heat shock proteins that minimize protein aggregation and stimulate protein refolding through intrinsic chaperone activities (3).The stress-induced modification of intracellular proteins by small ubiquitin-like modifier (SUMO) 1 has recently emerged as an additional line of defense in eukaryotes (4 -6). Attachment of the ϳ100-amino-acid SUMO protein is driven by an ATPdependent, three-step enzyme cascade, which in Arabidopsis thaliana involves the E1 heterodimer (SAE2 together with either of two SAE1 isoforms), a single E2 SCE1, and at least two E3s (SAP, MIZ1 (SIZ1), and MMS21/HYP2) (7-11). The end result is the isopeptide linkage of one or more SUMO moieties to accessible target lysine(s). Most commonly, a consensus ⌿KxE SUMO-binding motif is modified, where ⌿ represents a bulky hydrophobic residue (12, 13). In some cases, the bound SUMOs themselves are also substrates, which results in poly-SUMO chains decorating the target (14, 15). Once generated, SUMO conjugates can be disassembled by a family of deSUMOylating proteases that specifically cleave these isopeptide bonds, thus allowing SUMO to act reversibly (e.g. Refs. 7,[16][17][18].Imp...

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“…These novel types of fragmentation have emerged as valuable alternatives to the widely used collision-induced dissociation (CID) fragmentation. Although less popular than CID, these novel types of fragmentation have proven to be complementary alternatives for protein and peptide identifi cation and quantitation [46], for peptide de novo sequencing [47] and for the characterisation of PTMs [48].…”

Section: Emerging Types Of Fragmentation In Mass Spectrometrymentioning

confidence: 99%

Approaches for the study of cancer: towards the integration of genomics, proteomics and metabolomics

et al. 2011

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iTRAQ-based quantitative analysis of protein mixtures with large fold change and dynamic range

Cited by 63 publications

References 12 publications

iTRAQ-based protein profiling provides insights into the central metabolism changes driving grape berry development and ripening

iTRAQ-based protein profiling provides insights into the central metabolism changes driving grape berry development and ripening

Quantitative Proteomics Reveals Factors Regulating RNA Biology as Dynamic Targets of Stress-induced SUMOylation in Arabidopsis

Approaches for the study of cancer: towards the integration of genomics, proteomics and metabolomics

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