scholarly journals Interactions of Gliotoxin with Protein Cysteines

2021 ◽  
Author(s):  
◽  
Rana Fathizargaran
Keyword(s):  
Mass Spectrometry ◽  
Creatine Kinase ◽  
Mammalian Cells ◽  
Protein Modification ◽  
Sulfur Oxidation ◽  
Maldi Mass Spectrometry ◽  
Disulfide Bridge ◽  
Covalent Modification ◽  
Oxidation Products ◽  
Affinity Tag

<p>Gliotoxin is a secondary metabolite that is produced by several species of fungi, and is toxic to mammalian cells. It is immunosuppressive, affects antigen presentation by macrophages, and causes apoptosis of some cells. Gliotoxin is an epipolythiodioxopiperazine molecule and contains an internal disulfide bridge that is highly reactive and essential for its toxicity. Suggested mechanisms of action include modification of thiol groups of cysteine residues in target proteins by generating oxidative stress or through covalent modification. The goal of this project was to develop mass spectrometry methods to detect protein modification by gliotoxin. Creatine kinase was used as a model protein. The measured mass of creatine kinase from 45 spectra gave a mean of 42,944 ± 24 which was consistent with the predicted mass of creatine kinase. A tryptic digest of creatine kinase indicated ions consistent with the predicted masses of the four cys-containing peptides including abundant ions at m/z 794, 1130 and 2870 and an ion at low intensity at 4373. The reaction of creatine kinase with gliotoxin showed a time dependent reaction that after 14 h was consistent with formation of a gliotoxin adduct. Reduction of the product with dithiothreitol released creatine kinase. Analysis of the tryptic peptides using MALDI mass spectrometry indicated complex modification of cysteines possibly including formation of a mixed disulfide adduct, intramolecular disulfides of CK, and sulfur oxidation products. Further analysis using the ICAT (isotope-coded affinity tag) method suggested modification of Cys-254 and Cys-283 by gliotoxin. Preliminary experiments examined the effects of gliotoxin on HL-60 cells using ICAT. Proteins of gliotoxin-treated and untreated cells were labeled with Heavy and Light ICAT reagents. Potential ICAT pairs were detected in the mass spectrum as a preliminary search for proteins affected by gliotoxin. The results indicate that ICAT labeling should be an effective strategy for characterization of the protein targets of gliotoxin.</p>

scholarly journals Interactions of Gliotoxin with Protein Cysteines

2021 ◽  
Author(s):  
◽  
Rana Fathizargaran
Keyword(s):  
Mass Spectrometry ◽  
Creatine Kinase ◽  
Mammalian Cells ◽  
Protein Modification ◽  
Sulfur Oxidation ◽  
Maldi Mass Spectrometry ◽  
Disulfide Bridge ◽  
Covalent Modification ◽  
Oxidation Products ◽  
Affinity Tag

<p>Gliotoxin is a secondary metabolite that is produced by several species of fungi, and is toxic to mammalian cells. It is immunosuppressive, affects antigen presentation by macrophages, and causes apoptosis of some cells. Gliotoxin is an epipolythiodioxopiperazine molecule and contains an internal disulfide bridge that is highly reactive and essential for its toxicity. Suggested mechanisms of action include modification of thiol groups of cysteine residues in target proteins by generating oxidative stress or through covalent modification. The goal of this project was to develop mass spectrometry methods to detect protein modification by gliotoxin. Creatine kinase was used as a model protein. The measured mass of creatine kinase from 45 spectra gave a mean of 42,944 ± 24 which was consistent with the predicted mass of creatine kinase. A tryptic digest of creatine kinase indicated ions consistent with the predicted masses of the four cys-containing peptides including abundant ions at m/z 794, 1130 and 2870 and an ion at low intensity at 4373. The reaction of creatine kinase with gliotoxin showed a time dependent reaction that after 14 h was consistent with formation of a gliotoxin adduct. Reduction of the product with dithiothreitol released creatine kinase. Analysis of the tryptic peptides using MALDI mass spectrometry indicated complex modification of cysteines possibly including formation of a mixed disulfide adduct, intramolecular disulfides of CK, and sulfur oxidation products. Further analysis using the ICAT (isotope-coded affinity tag) method suggested modification of Cys-254 and Cys-283 by gliotoxin. Preliminary experiments examined the effects of gliotoxin on HL-60 cells using ICAT. Proteins of gliotoxin-treated and untreated cells were labeled with Heavy and Light ICAT reagents. Potential ICAT pairs were detected in the mass spectrum as a preliminary search for proteins affected by gliotoxin. The results indicate that ICAT labeling should be an effective strategy for characterization of the protein targets of gliotoxin.</p>

Rapid detection of apoptosis in mammalian cells by using intact cell MALDI mass spectrometry

The Analyst ◽  
2011 ◽  
Vol 136 (24) ◽  
pp. 5181 ◽  
Author(s):  
Hongjuan Dong ◽  
Wei Shen ◽  
Myra Ting Wai Cheung ◽  
Yimin Liang ◽  
Hon Yeung Cheung ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rapid Detection ◽  
Mammalian Cells ◽  
Intact Cell ◽  
Maldi Mass Spectrometry

scholarly journals Food Protein Sterylation: Chemical Reactions between Reactive Amino Acids and Sterol Oxidation Products under Food Processing Conditions

Foods ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1882
Author(s):  
Franks Kamgang Nzekoue ◽  
Thomas Henle ◽  
Giovanni Caprioli ◽  
Gianni Sagratini ◽  
Michael Hellwig
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Food Processing ◽  
Protein Modification ◽  
Unsaturated Fatty Acids ◽  
Oxidation Products ◽  
Charge Ratio ◽  
Array Detector ◽  
Food Protein ◽  
Secondary Products

Sterols, especially cholesterol and phytosterols, are important components of food lipids. During food processing, such as heating, sterols, like unsaturated fatty acids, can be oxidized. Protein modification by secondary products of lipid peroxidation has recently been demonstrated in food through a process called lipation. Similarly, this study was performed to assess, for the first time, the possibility of reactions between food proteins and sterol oxidation products in conditions relevant for food processing. Therefore, reaction models consisting of oxysterol (cholesterol 5α,6α-epoxide) and reactive amino acids (arginine, lysine, and methionine) were incubated in various conditions of concentration (0–8 mM), time (0–120 min), and temperature (30–180 °C). The identification of lysine adducts through thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) with a diode array detector (DAD), and electrospray ionization (ESI) mass spectrometry (MS) evidenced a reaction with lysine. Moreover, the HPLC-ESI with tandem mass spectrometry (MS/MS) analyses allowed observation of the compound, whose mass to charge ratio m/z 710.5 and fragmentation patterns corresponded to the reaction product [M + H]+ between cholesterol-5α,6α-epoxide and the ε-amino-group of Nα-benzoylglycyl-l-lysine. Moreover, kinetic studies between Nα-benzoylglycyl-l-lysine as a model for protein-bound lysine and cholesterol 5α,6α-epoxide were performed, showing that the formation of lysine adducts strongly increases with time, temperature, and oxysterol level. This preliminary study suggests that in conditions commonly reached during food processing, sterol oxidation products could react covalently with protein-bound lysine, causing protein modifications.

scholarly journals A combination of electrochemistry and mass spectrometry to monitor the interaction of reactive species with supported lipid bilayers

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
M. Ravandeh ◽  
H. Kahlert ◽  
H. Jablonowski ◽  
J.-W. Lackmann ◽  
J. Striesow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid Bilayers ◽  
Mammalian Cells ◽  
Reactive Species ◽  
Oxidation Products ◽  
Supported Lipid Bilayers ◽  
Resonance Spectroscopy ◽  
Membrane Composition ◽  
Physical Plasma ◽  
The Impact

Abstract Reactive oxygen and nitrogen species (RONS), e.g. generated by cold physical plasma (CPP) or photodynamic therapy, interfere with redox signaling pathways of mammalian cells, inducing downstream consequences spanning from migratory impairment to apoptotic cell death. However, the more austere impact of RONS on cancer cells remains yet to be clarified. In the present study, a combination of electrochemistry and high-resolution mass spectrometry was developed to investigate the resilience of solid-supported lipid bilayers towards plasma-derived reactive species in dependence of their composition. A 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer was undisturbed by 200 µM H2O2 (control) but showed full permeability after CPP treatment and space-occupying oxidation products such as PoxnoPC, PAzePC, and POPC hydroperoxide were found. Electron paramagnetic resonance spectroscopy demonstrated the presence of hydroxyl radicals and superoxide anion/hydroperoxyl radicals during the treatment. In contrast, small amounts of the intramembrane antioxidant coenzyme Q10 protected the bilayer to 50% and LysoPC was the only POPC derivative found, confirming the membrane protective effect of Q10. Such, the lipid membrane composition including the presence of antioxidants determines the impact of pro-oxidant signals. Given the differences in membrane composition of cancer and healthy cells, this supports the application of cold physical plasma for cancer treatment. In addition, the developed model using the combination of electrochemistry and mass spectrometry could be a promising method to study the effect of reactive species or mixes thereof generated by chemical or physical sources.

scholarly journals Unbiased Identification of Proteins Covalently Modified by Complex Mixtures of Peroxidized Lipids Using a Combination of Electrophoretic Mobility Band Shift with Mass Spectrometry

Antioxidants ◽  
2018 ◽  
Vol 7 (9) ◽  
pp. 116 ◽  
Author(s):  
Bernd Gesslbauer ◽  
David Kuerzl ◽  
Niko Valpatic ◽  
Valery Bochkov
Keyword(s):  
Oxidative Stress ◽  
Mass Spectrometry ◽  
Electrophoretic Mobility ◽  
Complex Mixture ◽  
Peptide Sequencing ◽  
Covalent Modification ◽  
Oxidation Products ◽  
Simple Method ◽  
Lipid Oxidation Products ◽  
Modified Proteins

Covalent modification of functionally important cell proteins by lipid oxidation products (LOPs) is a known mechanism initiating pathological consequences of oxidative stress. Identification of new proteins covalently modified by electrophilic lipids can be performed by a combination of chemical, immunological, and mass spectrometry-based methods, but requires prior knowledge either on the exact molecular structure of LOPs (e.g., 4-hydroxynonenal) or candidate protein targets. However, under the conditions of oxidative stress in vivo, a complex mixture of proteins (e.g., cytosolic proteome) reacts with a complex mixture of LOPs. Here we describe a method for detection of lipid-modified proteins that does not require an a priori knowledge on the chemical structure of LOPs or identity of target proteins. The method is based on the change of electrophoretic mobility of lipid-modified proteins, which is induced by conformational changes and cross-linking with other proteins. Abnormally migrating proteins are detected by mass spectrometry-based protein peptide sequencing. We applied this method to study effects of oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) on endothelial cells. Several known, but also many new, OxPAPC-binding proteins were identified. We expect that this technically relatively simple method can be widely applied for label-free analysis of lipid-protein interactions in complex protein samples treated with different LOPs.

Time-dependent cortisol turnover in tissues using stable isotope tracers and MALDI Mass Spectrometry (MS) sampling

2018 ◽  
Author(s):  
Shazia Khan ◽  
Diego F Cobice ◽  
Dawn EW Livingstone ◽  
C Logan Mackay ◽  
Scott P Webster ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Stable Isotope ◽  
Maldi Mass Spectrometry ◽  
Time Dependent ◽  
Stable Isotope Tracers ◽  
Isotope Tracers

Unrestrictive protein modification localization and quality control for open search of mass spectra

2018 ◽  
Author(s):  
Zhiwu An ◽  
Fuzhou Gong ◽  
Yan Fu
Keyword(s):  
Mass Spectrometry ◽  
Quality Control ◽  
Tandem Mass Spectrometry ◽  
Mass Spectra ◽  
Protein Modification ◽  
Software Tool ◽  
Tandem Mass ◽  
Simulation Data ◽  
Post Translational Modifications

We have developed PTMiner, a first software tool for automated, confident filtering, localization and annotation of protein post-translational modifications identified by open (mass-tolerant) search of large tandem mass spectrometry datasets. The performance of the software was validated on carefully designed simulation data. <br>

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