Albumin Is the Main Nucleophilic Target of Human Plasma: A Protective Role Against Pro-atherogenic Electrophilic Reactive Carbonyl Species?

Aldini, Giancarlo; Vistoli, Giulio; Regazzoni, Luca; Gamberoni, L.; Facino, Roberto Maffei; Yamaguchi, Satoru; Uchida, Koji

doi:10.1021/tx700349r

Cited by 100 publications

(109 citation statements)

References 61 publications

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“…8B). Physiological processes such as the regulation of vascular homeostasis, oxidative stress, and lipid peroxidation generate a wide range of small electrophiles that react with HSA-Cys 34 , including nitric oxide, small aldehydes, and oxygen radicals (18,30,31). Because the thiol of HSA-Cys 34 resides inside a 10 Å crevice in the HSA molecule (32,33), the preponderance of small added masses in our sample may also reflect steric restriction of bulky molecules from the reaction site.…”

Section: Fs-srm Of T3 Adducts From Human Samples-followingmentioning

confidence: 96%

“…Because we had previously observed that specific xenobiotic adducts of HSA-Cys 34 were present at much higher concentrations than those of Hb-Cys 93␤ in human blood (14 -16), we chose HSA-Cys 34 for profiling experiments. In fact, HSA-Cys 34 is a well known scavenger of small electrophiles that accounts for about 80% of all free thiols in human serum (17,18), and HSA-Cys 34 adducts have been unambiguously detected from reactions with aldehydes, nitrogen mustards, oxiranes, quinones, metal ions, and a host of drugs [reviewed in (7)]. Following tryptic digestion of HSA, all thiol adducts should be bound to a single peptide, which we designate as T3 (the third largest tryptic peptide of HSA), with sequence ALVLIAFAQYLQQC 34 PFEDHVK and an average mass of 2433.87 Da.…”

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confidence: 99%

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Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

Grigoryan

Funk

et al. 2011

Molecular & Cellular Proteomics

118

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A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys 34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSACys 34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys 34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys 34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ؎2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected band y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.004606, 1-13, 2011.Reactive electrophiles, particularly aldehydes, epoxides, quinones, and short-lived oxygen and nitrogen species, have long been implicated as causes of major human diseases (1-5). These electrophiles enter the blood following metabolism of exogenous compounds, from oxidation of lipids and other natural molecules, from ionizing radiation, and from inflammation associated with infections and disease processes. Because of their short life spans in vivo, it is rarely possible to measure reactive electrophiles in body fluids. However, adducts produced by reactions with blood nucleophiles, notably hemoglobin (Hb) 1 and human serum albumin (HSA), reflect the presence of reactive electrophiles in the systemic circulation (6, 7). Levels of targeted Hb and/or HSA adducts have been investigated in human blood for several exogenous or endogenous toxicants that are either electrophilic carcinogens or their precursors, notably, acrylamide, aflatoxin B1, benzene, 1,3-butadiene, ethylene oxide, as well as assorted aromatic amines, polycyclic aromatic hydrocarbons, and aldehyde...

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Section: Fs-srm Of T3 Adducts From Human Samples-followingmentioning

confidence: 96%

mentioning

confidence: 99%

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

Grigoryan

Funk

et al. 2011

Molecular & Cellular Proteomics

118

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“…On the basis of these findings, we proposed [59] a new MS approach, based on LC-MS/MS analysis of tag HNE/ACR-modified peptides of carbonylated albumin and actin that can be applied to the digested proteins isolated from the biological samples or directly to the digested sample (one-shot procedure). By using 2-D SDS-PAGE followed by immunoblot analysis and MALDI-TOF mass fingerprint analysis of the isolated and digested protein, we demonstrated that HSA is the major nucleophilic plasma target of HNE [60] and that Cys 34 is the target site on HSA for a,b-unsaturated aldehydes adduction.…”

Section: In Vitro Evidencementioning

confidence: 98%

Protein modification by acrolein: Relevance to pathological conditions and inhibition by aldehyde sequestering agents

Aldini

Orioli

Carini

2011

Molecular Nutrition Food Res

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Acrolein (ACR) is a toxic and highly reactive α,β-unsaturated aldehyde widely distributed in the environment as a common pollutant and generated endogenously mainly by lipoxidation reactions. Its biological effects are due to its ability to react with the nucleophilic sites of proteins, to form covalently modified biomolecules which are thought to be involved as pathogenic factors in the onset and progression of many pathological conditions such as cardiovascular and neurodegenerative diseases. Functional impairment of structural proteins and enzymes by covalent modification (crosslinking) and triggering of key cell signalling systems are now well-recognized signs of cell and tissue damage induced by reactive carbonyl species (RCS). In this review, we mainly focus on the in vitro and in vivo evidence demonstrating the ability of ACR to covalently modify protein structures, in order to gain a deeper insight into the dysregulation of cellular and metabolic pathways caused by such modifications. In addition, by considering RCS and RCS-modified proteins as drug targets, this survey will provide an overview on the newly developed molecules specifically tested for direct or indirect ACR scavenging, and the more significant studies performed in the last years attesting the efficacy of compounds already recognized as promising aldehyde-sequestering agents.

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“…We have previously found that free plasma RCSs rapidly disappear (by almost 70% within 5 min) during reperfusion, since they form covalent adducts with plasma proteins [22]. By proteomic studies, we identified albumin as their main protein target and Cys34 as the most reactive site, whose high nucleophilic reactivity has been clarified by molecular modeling studies [35]. Based on these results, we aimed at identifying the chemical structures of RCSs adducted to Cys34.…”

Section: Discussionmentioning

confidence: 97%

Albumin Cys34 adducted by acrolein as a marker of oxidative stress in ischemia-reperfusion injury during hepatectomy

Witort

Capaccioli

Becatti

et al. 2016

Free Radical Research

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The aim of this study was to measure and identify the reactive carbonyl species (RCSs) released in the blood of humans subjected to hepatic resection. Pre-anesthesia malondialdehyde (MDA) plasma content (0.36 ± 0.11 nmol/mg protein) remained almost unchanged immediately after anaesthesia, before clamping and at the 10th min after ischemia, while markedly increased (to 0.59 ± 0.07 nmol/mg; p < 0.01, Tukey's post test) at the 10th min of reperfusion. A similar trend was observed for the protein carbonyls (PCs), whose pre-anesthesia levels (0.17 ± 0.13 nmol/mg) did not significantly change during ischemia, while increased more than fourfold at the 10th min of reperfusion (0.75 ± 0.17 nmol/mg; p < 0.01, Tukey's post test). RCSs were then identified as covalent adducts to the albumin Cys34, which we previously found as the most reactive protein nucleophilic site in plasma. By using a mass spectrometry (MS) approach based on precursor ion scanning, we found that acrolein (ACR) is the main RCS adducted to albumin Cys34. In basal conditions, the adducted albumin was 0.6 ± 0.4% of the native form but it increased by almost fourfold at the 10th min of reperfusion (2.3 ± 0.7%; p < 0.01, t-test analysis). Since RCSs are damaging molecules, we propose that RCSs, and ACR in particular, are new targets for novel molecular treatments aimed at reducing the ischemia/reperfusion damage by the use of RCS sequestering agents. ARTICLE HISTORY

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Albumin Is the Main Nucleophilic Target of Human Plasma: A Protective Role Against Pro-atherogenic Electrophilic Reactive Carbonyl Species?

Cited by 100 publications

References 61 publications

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

Protein modification by acrolein: Relevance to pathological conditions and inhibition by aldehyde sequestering agents

Albumin Cys34 adducted by acrolein as a marker of oxidative stress in ischemia-reperfusion injury during hepatectomy

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