Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin–fluorescein isothiocyanate conjugates

Jacksén, Johan; Dahl, Kenneth; Karlberg, Ann‐Therése; Redeby, Theres; Emmer, Åsa

doi:10.1016/j.jchromb.2010.03.024

Cited by 4 publications

(3 citation statements)

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“…Additionally, the presence of IgG and IgM antibodies against xenobiotics conjugated to human serum Alb in a study of serum from healthy donors, suggests that Alb conjugates can activate the immune system ( Vojdani et al, 2015 ). The adductome of other skin proteins with haptens, such as human cytokeratin 14 and human cofilin ( Aleksic et al, 2008 ), as well as protein lysates from keratinocyte cell lines and human skin tissue ( Jacksén et al, 2010 ; Simonsson et al, 2011 ; Parkinson et al, 2018 ; Parkinson et al, 2020b ; Bailey et al, 2021 ) has also been investigated. Despite the extensive mapping of the most reactive sites of relevant proteins, no protein-conjugates have been identified in vivo except the TRITC-MIF conjugate identified by us ( Karlsson et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Haptenation of Macrophage Migration Inhibitory Factor: A Potential Biomarker for Contact Hypersensitivity

et al. 2022

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The immunological response in contact hypersensitivity is incited by small electrophilic compounds, known as haptens, that react with endogenous proteins after skin absorption. However, the identity of hapten-modified proteins seen as immunogenic remains as yet largely unknown. In a recent study, we have for the first time identified a hapten-modified protein in the local lymph nodes of mice treated topically with the model hapten tetramethylrhodamine isothiocyanate (TRITC). The TRITC modification was located on the N-terminal proline of the protein macrophage migration inhibitory factor (MIF). The focus of the current study was to investigate the presence of the same hapten-protein conjugate in blood samples from mice treated topically with TRITC. Furthermore, TRITC modifications of the two major blood proteins, namely hemoglobin (Hb) and albumin (Alb), as well as TRITC modifications of MIF other than the N-terminal proline, were examined. Following incubation with different molar ratios of TRITC, a proteomic approach was applied to characterize conjugate formation of the three aforementioned proteins, using high resolution mass spectrometry (HRMS). The targeted screening of the TRITC-treated mice blood and lymph node samples for these sites led to the identification of only the same TRITC-MIF conjugate previously detected in the lymph nodes. No Hb and Alb conjugates were detected. Quantification of both the TRITC-modified and unmodified N-terminal peptide of MIF in blood and lymph node samples gave interesting insights of MIF’s role in murine contact hypersensitivity. Incubation of MIF with four different haptens encompassing different reactivity mechanisms and potencies, showed adduct formation at different amino acid residues, suggesting that MIF can be the preferred target for a wide variety of haptens. The present study provides essential progress toward understanding of hapten-protein conjugate formation in contact hypersensitivity and identifies hapten-modified MIF as a potential biomarker for this condition. Further investigation of MIF as a target protein can be a next step to determine if MIF is a biomarker that can be used to develop better diagnostic tools and targeted therapeutics for individuals with allergic contact dermatitis.

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Section: Introductionmentioning

confidence: 99%

Haptenation of Macrophage Migration Inhibitory Factor: A Potential Biomarker for Contact Hypersensitivity

et al. 2022

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“…FITC labelled BSA (FITC-BSA) is a commonly used fluorescent marker, and the average degree of substitution has been determined by absorbance and fluorescence spectroscopy, gel electrophoresis, size exclusion and ion exchange chromatography or capillary isotachophoresis [31] [32] [33] [34].…”

Section: Introductionmentioning

confidence: 99%

An investigation of paper based microfluidic devices for size based separation and extraction applications

Zhong

Wang

et al. 2015

Journal of Chromatography B

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“…Despite the important impact of labeling on the performance of fluorescent studies, protein labeling is usually carried out with no particular optimization by letting the reagents react for several hours or overnight using empirical protocols. [3,4] A few reports have studied the kinetics of labeling, but used complex techniques such as Maldi-TOF [5,6] and capillary electrophoresis. [7,8] There is therefore a need for a simple method that allows the generic monitoring of labeling reactions.…”

Section: Introductionmentioning

confidence: 99%

Monitoring Labeling Reactions Using Fluorescence Polarization

Crittin

Héritier

Segura³

2013

Chimia

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Fluorescence techniques are widely applied in protein research owing to their specificity and sensitivity, but require prior fluorescent labeling. Here we show a novel approach to optimize labeling protocols by monitoring labeling reactions using fluorescence polarization: the larger molecular mass of the fluorescent protein conjugate compared to the dye alone results in an increase in fluorescence anisotropy during the reaction. Thereby, labeling of lysozyme with fluorescein isothiocyanate or carboxyfluorescein succinimidyl ester could be monitored and the influence of parameters such as the pH could be quantitatively assessed. Moreover, the impact and kinetics of side reactions such as hydrolysis were determined. This new method is rapid, easy to implement, and generically applicable.

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Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin–fluorescein isothiocyanate conjugates

Cited by 4 publications

References 31 publications

Haptenation of Macrophage Migration Inhibitory Factor: A Potential Biomarker for Contact Hypersensitivity

Haptenation of Macrophage Migration Inhibitory Factor: A Potential Biomarker for Contact Hypersensitivity

An investigation of paper based microfluidic devices for size based separation and extraction applications

Monitoring Labeling Reactions Using Fluorescence Polarization

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