Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

Marie, Anne-Lise; Przybylski, Cédric; Gonnet, Florence; Daniel, Régis; Urbain, Rémi; Chevreux, Guillaume; Jorieux, Sylvie; Taverna, Myriam

doi:10.1016/j.aca.2013.09.023

Cited by 39 publications

(26 citation statements)

References 32 publications

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“…Due to the low concentrations of free thiols in plasma, albumin may well be the main thiol antioxidant in circulation (76,94). This can undergo oxidation to form a mixed disulfide with cysteine (cysteinylation), homocysteine (32) or a sulfinic acid (66). Increased levels of cysteinylated albumin have been associated with intrauterine growth restriction (3).…”

Section: Oxidized Proteins In Circulationmentioning

confidence: 99%

Redox Proteomics Applied to the Thiol Secretome

Ghezzi

Chan

2017

Antioxidants & Redox Signaling

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Word count: 6800 excluding references Number of references: 113Number of greyscale and color illustrations: 7 greyscale and 5 color (online only)Ghezzi 2 AbstractStudies of redox proteomics to identify glutathionylated proteins have so far been focused on intracellular proteins. However, plasma proteins have been described as glutathionylated. The present review discusses the redox state of protein cysteines in relation to their cellular distribution. We focus in particular on the redox state of proteins secreted under inflammatory conditions and that may act as soluble mediators (cytokines). We describe the various approaches used to detect secreted glutathionylated proteins, the only thiol modification studied so far in secreted proteins, and the specific problems presented in the study of the secretome. This review will deal with a particular aspect of redox proteomics, the analysis of secreted proteins and their possible function. Ghezzi 3 Redox state of proteins in the context of their localization Redox proteomics of the thiol proteomeAccording to the Oxford English Dictionary, proteomics is "the study or analysis of the set of proteins expressed by an organism" and we commonly apply the term to high-throughput techniques of protein identification. As such, the history of redox proteomics started in 2002 when various groups published the first reports of the identification of oxidized proteins by modifying the existing proteomic techniques.Two of these papers were aimed at carbonylated proteins (19,20) and others at protein glutathionylation (33,37,61) or overall protein thiol oxidation (60) .This review will deal with the redox states of protein thiols. Biochemistry textbooks and protein databases classify cysteines in proteins as either free or engaged in a disulfide bond. However, "free" cysteines can be reversibly or irreversibly oxidized to various forms. In particular, in addition to forming structural disulfides, cysteines can form transient, reversible disulfides with another protein cysteine, with free cysteine (cysteinylation) or with the cysteine in the tripeptide glutathione (GSH;; glutamylcystenyl-glycine). Fig. 1 lists the most important oxidation state of sulfur in cysteine.These forms of oxidation, due to their reversibility, are of great interest in the context of redox regulation, as a means by which the redox state of the cell can regulate protein functions (43-45).Specific proteins undergoing glutathionylation were described even before the term "proteomics" became popular (in the late 1990s, according to Google books ngram viewer -https://books.google.com/ngrams). Two proteins have been identified as undergoing glutathionylation in two separate papers, an unidentified 30 kDa cytosolic Ghezzi 4protein in oxidant-treated rat liver (79), and actin in human neutrophils during oxidative burst (22). Although these were not the result of high-throughput studies, some of the technologies used were then adapted for two-dimensional gels for further studies and are at the basis of current red...

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Section: Oxidized Proteins In Circulationmentioning

confidence: 99%

Redox Proteomics Applied to the Thiol Secretome

Ghezzi

Chan

2017

Antioxidants & Redox Signaling

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“…The effective and total lengths were 50 and 60.2 cm, respectively. New fused‐silica capillaries were coated with PEO according to the procedure previously described . Briefly, the capillary was rinsed with water (5 min), 0.1 N NaOH (5 min), 1 N HCl (10 min), water (10 min), 0.2% w/v PEO solution in 0.1 N HCl (5 min), and water (5 min).…”

Section: Methodsmentioning

confidence: 99%

A capillary zone electrophoresis method to detect conformers and dimers of antithrombin in therapeutic preparations

et al. 2016

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Antithrombin (AT) is a human plasma glycoprotein that possesses anticoagulant and anti-inflammatory properties. However, the native (active) form of AT is unstable and undergoes conformational changes, leading to latent, cleaved, and heterodimeric forms. The presence of these alternative forms mostly inactive can highly impact the quality and therapeutic activity of pharmaceutical AT preparations. We developed a capillary zone electrophoresis method, based on a neutral polyethylene oxide-coated capillary and a buffer close to physiological conditions, enabling the separation of more than eight forms of AT. Several peaks were identified as native, latent, and heterodimeric forms. The CZE method was reproducible with intraday relative standard deviations less than 0.5 and 2% for migration times and peak areas, respectively. The method was applied to the comparison of AT preparations produced by five competitive pharmaceutical companies, and statistical tests were performed. Important differences in the proportion of each form were highlighted. In particular, one AT preparation was shown to contain a high quantity of heterodimer, and two preparations contained high quantities of latent form. In addition, one AT preparation exhibited additional forms, not yet identified.

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“…It is simple, easy-to-use, and requires minimal reagents compared to chromatographic methods. Additionally, in CZE, the separation of analytes is based on their size-to-charge ratio making it well suited for separations of proteins with post-translational modifications (PTMs) or degradations that affect the charge of the molecule [6, 7] including deamidation, glycosylation, and phosphorylation.…”

Section: Techniquesmentioning

confidence: 99%

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

2014

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The development of therapeutic proteins and peptides is an expensive and time-intensive process. Biologics, which have become a multi-billion dollar industry, are chemically complex products that require constant observation during each stage of development and production. Post-translational modifications along with chemical and physical degradation from oxidation, deamidation, and aggregation, lead to high levels of heterogeneity that affect drug quality and efficacy. The various separation modes of capillary electrophoresis (CE) are commonly utilized to perform quality control and assess protein heterogeneity. This review attempts to highlight the most recent developments and applications of CE separation techniques for the characterization of protein and peptide therapeutics by focusing on papers accepted for publication in the in the two-year period between January 2012 and December 2013. The separation principles and technological advances of CE, capillary gel electrophoresis, capillary isoelectric focusing, capillary electrochromatography and CE-mass spectrometry are discussed, along with exciting new applications of these techniques to relevant pharmaceutical issues. Also included is a small selection of papers on microchip electrophoresis to show the direction this field is moving with regards to the development of inexpensive and portable analysis systems for on-site, high-throughput analysis.

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Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

Cited by 39 publications

References 32 publications

Redox Proteomics Applied to the Thiol Secretome

Redox Proteomics Applied to the Thiol Secretome

A capillary zone electrophoresis method to detect conformers and dimers of antithrombin in therapeutic preparations

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

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