The Comparison of Plasma Deproteinization Methods for the Detection of Low-Molecular-Weight Metabolites by 1H Nuclear Magnetic Resonance Spectroscopy

Daykin, Clare A.; Foxall, P.J.D.; Connor, Susan C.; Lindon, John C.; Nicholson, Jeremy K.

doi:10.1006/abio.2002.5637

Cited by 141 publications

(110 citation statements)

References 17 publications

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“…First, proteins can bind many small molecules such as citrate, lactate, aromatic amino acid, and metabolites, shortening their relaxation time T 2 to that of the protein. Hence, the use of CPMG [29], optimized for the protein signal filtering, leads (in the most favorable cases) to underestimating the concentrations of some small molecules or (in the worst cases) to completely missing the analyte (e.g., seeing the phenol red). Indeed, phenol red signal, a sharp doublet at 6.7 ppm in DMEM without serum (Fig.…”

Section: The Serum Issue In Q-nmrmentioning

confidence: 99%

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Goldoni

Beringhelli

Rocchia

et al. 2016

Analytical Biochemistry

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a b s t r a c tAbsolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] ¼ 5À25 mM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (CarrePurcell eMeiboomeGill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction.© 2016 Elsevier Inc. All rights reserved.Interest in quantitative nuclear magnetic resonance (q-NMR) analysis has steadily increased since the seminal works of Jungnickel and Forbes [1] and Hollis [2], owing to the unique ability of this methodological approach to quantify, at the same time, a wide range of structurally diverse biological substances with high throughput and automation [3]. Compared with other analytical techniques, q-NMR does not require chromatographic separation, generates signals that are directly proportional to the number of NMR-active nuclei in the targeted analyte [4,5], and offers a high degree of assay reproducibility [6] along with reduced uncertainty [7,8]. Moreover, advances in hardware developmentdsuch as the introduction of high-field magnets and cryogenic probesdhave progressively lowered the limit of detection (LOD) for analytes, thereby improving the overall sensitivity of the technique [4].The PULCON (pulse length-based concentration determination) procedure [9], one of the most promising methods for q-NMR [4], finds its mathematical and physical foundation in the principle of reciprocity [10e12]. This principle states that the strength of a signal is inversely proportional to the 90 pulse length in the active volume of the NMR tube. The PULCON method can be implemented in all types of commercial NMR spectrometers and requires only one Abbreviations used: q-NMR, quantitative nuclear magnetic resonance; LOD, limit of detection; PULCON, pulse length-based concentration determination; CPMG,

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Section: The Serum Issue In Q-nmrmentioning

confidence: 99%

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Goldoni

Beringhelli

Rocchia

et al. 2016

Analytical Biochemistry

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“…The results of this test are not far from those taken from the electrophoretic analysis. This is expected because the albumin is the dominant protein in plasma [3]. In conclusion, acetone and acetonitrile are much better than methanol and ethanol for protein elimination from plasma samples.…”

Section: Protein Determinationmentioning

confidence: 83%

“…This should improve the MS sensitivity and avoid instrument capillary blockage. In addition, solvent deproteination has the advantage of better analyte recovery over the other methods [3,4]. In case of urine samples, some researchers prefer to reduce the salt in samples by further diluting them with water or with any water-miscible solvent [10].…”

Section: Introductionmentioning

confidence: 99%

“…In addition to removing proteins and salts from biological samples, we have found during our metabonomic studies that it extraction, which are expensive and low-recovery deproteinais also often essential to remove phospholipids or to reduce their tion procedures [3], precipitation of proteins from serum or original concentrations because their strong signals may obscure plasma samples can be carried out by adding salts, some acids other more important biomarkers and they tend to appear as background peaks throughout the chromatogram. In addition, they lack the reproducible responses required in MS analysis by nuclear magnetic resonance (NMR) spectroscopy, addition of [11].…”

Section: Introductionmentioning

confidence: 99%

“…or water-miscible organic solvents [3][4][5][6][7][8][9]. Although all of these procedures can be used in discarding proteins prior to analysis…”

Section: Introductionmentioning

confidence: 99%

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Comparison of different water-miscible solvents for the preparation of plasma and urine samples in metabolic profiling studies

Alzweiri

Watson

Robertson

et al. 2008

Talanta

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This version is available at https://strathprints.strath.ac.uk/7602/ Strathprints is designed to allow users to access the research output of the University of Strathclyde. Unless otherwise explicitly stated on the manuscript, Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Please check the manuscript for details of any other licences that may have been applied. You may not engage in further distribution of the material for any profitmaking activities or any commercial gain. You may freely distribute both the url (https://strathprints.strath.ac.uk/) and the content of this paper for research or private study, educational, or not-for-profit purposes without prior permission or charge.Any correspondence concerning this service should be sent to the AbstractThe Lowry method and a capillary electrophoresis method were used to analyse protein residues in the supernatant after solvent deproteination of plasma. Acetonitrile and acetone were much more effective than methanol and ethanol at reducing the levels of proteins in plasma. The ability of different solvents to decrease levels of phospholipids in plasma samples was assessed using electrospray ionisation mass spectrometry (MS). Phospholipid signals can obscure differences between samples in general metabolite profiling (i.e. non-target compound) studies. Acetonitrile was much more effective than methanol in reducing the MS signal due to phospholipids in plasma which is a consequence of the poor solubility of phospholipids in acetonitrile. The capability of the solvents at reducing salts in urine samples was also studied by using an amperometric method. Using this approach little difference was detected between methanol, ethanol, acetonitrile and acetone in their ability to desalt urine samples.

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An FITC‐BODIPY FRET Couple: Application to Selective, Ratiometric Detection and Bioimaging of Cysteine

Hee

Kim

Akisawa

et al. 2014

Chemistry An Asian Journal

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A novel FRET couple of fluorescein is disclosed, and it was readily constructed by conjugating an amino-BODIPY dye, a new FRET donor, with fluorescein isocyanate. Its potential was demonstrated by a fluorescence sensing system for cysteine, which was prepared by introducing acryloyl groups to the fluorescein moiety. The FRET probe exhibited promising ratiometric response to cysteine with high selectivity and sensitivity in a buffer solution containing acetonitrile at a physiological pH of 7.4, but showed slow reactivity. This slow response was solved by addition of a surfactant, thus allowing ratiometric imaging and determination of the endogenous level of cysteine in cells in HEPES buffer, by confocal fluorescence microscopy. Imaging experiments toward various cells suggested that such aryl acrylate type probes are vulnerable to the ubiquitous esterase activity. For the selected C6 cell line, in which the esterase activity was minimal, the ratiometric quantification of cysteine level was demonstrated. The FRET probe was also applied to determine the level of cysteine in human blood plasma.

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The Comparison of Plasma Deproteinization Methods for the Detection of Low-Molecular-Weight Metabolites by 1H Nuclear Magnetic Resonance Spectroscopy

Cited by 141 publications

References 17 publications

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Comparison of different water-miscible solvents for the preparation of plasma and urine samples in metabolic profiling studies

An FITC‐BODIPY FRET Couple: Application to Selective, Ratiometric Detection and Bioimaging of Cysteine

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