Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction
Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use...
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| Published in | Analytical and bioanalytical chemistry Vol. 395; no. 3; pp. 809 - 817 |
|---|---|
| Main Authors | , , , |
| Format | Journal Article |
| Language | English |
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Berlin/Heidelberg
Berlin/Heidelberg : Springer-Verlag
01.10.2009
Springer-Verlag Springer |
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| Abstract | Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-¹³C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 μM for glutathione, 0.440 μM for phytochelatin 2, and 0.120 μM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation. [graphic removed] |
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| AbstractList | Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-¹³C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 μM for glutathione, 0.440 μM for phytochelatin 2, and 0.120 μM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation. [graphic removed] Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine- 13 C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 μM for glutathione, 0.440 μM for phytochelatin 2, and 0.120 μM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation. Figure This figure was created using Adobe Photoshop CS3 (San Jose, CA). It displays an image of a chromatogram from our manuscript (a phytochelatin extraction) overlaid upon an image of Chlorella vulgaris and laboratory equipment and materials used in our methods. Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 *mM for glutathione, 0.440 *mM for phytochelatin 2, and 0.120 *mM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation. Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-(13)C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 microM for glutathione, 0.440 microM for phytochelatin 2, and 0.120 microM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation.Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-(13)C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 microM for glutathione, 0.440 microM for phytochelatin 2, and 0.120 microM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation. Phytochelatins are short, cysteine-containing, detoxification peptides produced by plants, algae, and fungi in response to heavy metal exposure. These peptides auto-oxidize easily. Current extraction protocols do not adequately address losses of phytochelatins because of their oxidation and the use of indirect methods for quantification. Method enhancements include the use of an argon environment during extraction to reduce auto-oxidation, the use of glycine-(13)C2-labeled glutathione as an internal standard, and an electrospray ionization source with a triple quadrupole mass spectrometer as a detector. The method-detection limits were 0.081 microM for glutathione, 0.440 microM for phytochelatin 2, and 0.120 microM for phytochelatin 3. These detection limits were comparable to similar studies and were not compromised incorporating these adjustments. The use of a labeled internal standard and an inert gaseous environment during sample preparation greatly improved calibration linearity and sensitivity. Furthermore, phytochelatin degradation was significantly reduced and more accurately tracked. Previous studies involving phytochelatin analyses have likely been subject to higher variability caused by this propensity for phytochelatins to degrade rapidly in air. The method adjustments were simple and cost-effective and allowed phytochelatin analyses to be performed for hours at a time with minimal auto-oxidation. |
| Author | Simmons, Denina B. D Hayward, Allison R Emery, R. J. Neil Hutchinson, Thomas C |
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| Keywords | Phytochelatins ESI-MS/MS HPLC-MS Internal standard Auto-oxidation Cysteine Peptides HPLC chromatography Glycine Electrospray Quadrupole spectrometer Sample preparation Indirect method Detection limit Oxidation Argon Quantitative analysis Glutathione Oxygen Algae Chlorella vulgaris Air Calibration Chemical ionization Heavy metal Reversed phase chromatography Sensitivity Linearity Mass spectrometry MS/MS Environment Mass spectrometry |
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| SubjectTerms | Adjustment air analysis Analytical Chemistry Applied sciences argon (noble gases) Auto-oxidation Biochemistry Characterization and Evaluation of Materials Chemistry Chemistry and Materials Science Chlorella vulgaris Chlorella vulgaris - chemistry Chromatographic methods and physical methods associated with chromatography Chromatography, High Pressure Liquid Chromatography, High Pressure Liquid - methods Cost analysis cost effectiveness Degradation detection limit ESI-MS/MS Exact sciences and technology Extraction Food Science fungi Global environmental pollution Glutathione Glutathione - analysis Glutathione - isolation & purification heavy metals HPLC-MS Internal standard ionization isolation & purification Laboratory Medicine Linearity methods Monitoring/Environmental Analysis Original Paper Other chromatographic methods oxidation Peptides phytochelatins Phytochelatins - chemistry Phytochelatins - isolation & purification Pollution reversed-phase high performance liquid chromatography Sensitivity and Specificity spectrometers Spectrometric and optical methods Spectrometry, Mass, Electrospray Ionization Spectrometry, Mass, Electrospray Ionization - methods Tandem Mass Spectrometry Tandem Mass Spectrometry - methods |
| Title | Identification and quantification of glutathione and phytochelatins from Chlorella vulgaris by RP-HPLC ESI-MS/MS and oxygen-free extraction |
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