Characterization and quantification of thiol-peptides in Arabidopsis thaliana using combined dilution and high sensitivity HPLC-ESI-MS-MS

Bellini, Erika; Borsò, Marco; Betti, Camilla; Bruno, Laura; Andreucci, Andrea; Castiglione, Monica Ruffini; Toppi, Luigi Sanità di

doi:10.1016/j.phytochem.2019.05.007

Cited by 20 publications

(20 citation statements)

References 22 publications

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“…In brief, the reaction mixture (100 μl) containing 200 mM HEPES–NaOH (pH 8.0), 10 mM 2-mercaptoethanol, 12.5 mM GSH, 100 μM Cd or 200 μM Zn, and 50 ng of recombinant PCS was incubated at 35 °C for 60 min, then terminated by the addition of 25 μl of 10% trifluoroacetic acid (TFA). The terminated reactions were maintained at 10 °C in the autosampler tray and immediately analyzed by HPLC-ESI-MS-MS to identify and quantify PC n produced using the analytical method described in ( Bellini et al , 2019 ). For an accurate quantification, terminated reactions were diluted by a factor of 100 only for PC 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Marchantia polymorpha and A. thaliana samples, previously stored at –80 °C, were extracted according to Bellini et al (2019) , whereas yeast cells were extracted following the protocol described in Ramos et al (2008) with some modifications. Briefly, yeast cells were resuspended in 300 μl of the extraction buffer containing 0.1% (v/v) TFA, 0.5 mM DTPA (diethylenetriaminepentaacetic acid), and 200 ng ml –1 (glycine- 13 C 2 , 15 N)-labeled GSH and PC 2 internal standards.…”

Section: Methodsmentioning

confidence: 99%

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Ancestral function of the phytochelatin synthase C-terminal domain in inhibition of heavy metal-mediated enzyme overactivation

Barbaro

Bellini

et al. 2020

Journal of Experimental Botany

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Phytochelatin synthases (PCSs) play essential roles in detoxification of a broad range of heavy metals in plants and other organisms. Until now, however, no PCS gene from liverworts, the earliest branch of land plants and possibly the first one to acquire a PCS with a C-terminal domain, has been characterized. In this study, we isolated and functionally characterized the first PCS gene from a liverwort, Marchantia polymorpha (MpPCS). MpPCS is constitutively expressed in all organs examined, with stronger expression in thallus midrib. The gene expression is repressed by Cd2+ and Zn2+. The ability of MpPCS to increase heavy metal resistance in yeast and to complement cad1-3 (the null mutant of the Arabidopsis ortholog AtPCS1) proves its function as the only PCS from M. polymorpha. Site-directed mutagenesis of the most conserved cysteines of the C-terminus of the enzyme further uncovered that two twin-cysteine motifs repress, to different extents, enzyme activation by heavy metal exposure. These results highlight an ancestral function of the PCS elusive C-terminus as a regulatory domain inhibiting enzyme overactivation by essential and non-essential heavy metals. The latter finding may be relevant for obtaining crops with decreased root to shoot mobility of cadmium, thus preventing its accumulation in the food chain.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Ancestral function of the phytochelatin synthase C-terminal domain in inhibition of heavy metal-mediated enzyme overactivation

Barbaro

Bellini

et al. 2020

Journal of Experimental Botany

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“…Each sample was extracted as described in Bellini et al (2019) [50]. Briefly, gametophytes were homogenized by a mixer mill (MM200, Retsch) with 2 agate grinding balls (5 mm diameter) at a vibrational frequency of 30 Hz for 2 min.…”

Section: γ-Ec Gsh and Pcn Extraction Characterization And Quantifmentioning

confidence: 99%

“…Thiol peptides (γ-EC, GSH, and PCn) were analyzed with an 1290 Infinity UHPLC (Agilent, Santa Clara, CA, USA), equipped with a thermostated autosampler, a binary pump, and a column oven, coupled to an API 4000 triple quadrupole mass spectrometer (AB Sciex, Concord, ON, Canada), equipped with a Turbo-V ion spray source (AB Sciex). Chromatographs were separated by a reverse-phase Phenomenex (Torrance, CA, USA) Kinetex 2.6 µm XB-C18 100 Å, 100 × 3 mm HPLC column, protected by a C18 3-mm ID security guard ULTRA cartridge, as described in Bellini et al (2019) [50]. The separation was achieved by means of a gradient solvent system (solvent A, acetonitrile with 0.1% (v/v) formic acid; solvent B, water with 0.1% (v/v) formic acid) as follows: solvent A was set at 2% for 5 min, raised with a linear gradient to 44% in 4.5 min, and then raised with a linear gradient to 95% in 1 min.…”

Section: γ-Ec Gsh and Pcn Extraction Characterization And Quantifmentioning

confidence: 99%

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The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins

Bellini

Maresca

Betti

et al. 2020

IJMS

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In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.

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Application of peptide biomarkers in life analysis based on liquid chromatography–mass spectrometry technology

Han

Cong

et al. 2022

BioFactors

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Biomedicine is developing rapidly in the 21st century. Among them, the qualitative and quantitative analysis of peptide biomarkers is of considerable importance for the diagnosis and therapy of diseases and the quality evaluation of drugs and food. The identification and quantitative analysis of peptides have been going on for decades. Traditionally, immunoassays or biological assays are generally used to quantify peptides in biological matrices. However, the selectivity and sensitivity of these methods cannot meet the requirements of the application. The separation and analysis technique of liquid chromatography-mass spectrometry (LC-MS) supplies a reliable alternative. In contrast to immunoassays, LC-MS methods are capable of providing the analytical prowess necessary to satisfy the demands of peptide biomarker research in the life sciences arena. This review article provides a historical account of the in-roads made by LC-MS technology for the detection of peptide biomarkers in the past 10 years, with the focus on the qualification/quantification developments and their applications.

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Characterization and quantification of thiol-peptides in Arabidopsis thaliana using combined dilution and high sensitivity HPLC-ESI-MS-MS

Cited by 20 publications

References 22 publications

Ancestral function of the phytochelatin synthase C-terminal domain in inhibition of heavy metal-mediated enzyme overactivation

Ancestral function of the phytochelatin synthase C-terminal domain in inhibition of heavy metal-mediated enzyme overactivation

The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins

Application of peptide biomarkers in life analysis based on liquid chromatography–mass spectrometry technology

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