Mara Colzani

Mass spectrometric approaches for the identification and quantification of reactive carbonyl species protein adducts

Our current knowledge of the occurrence of proteins covalently modified by reactive carbonyl species (RCS) generated by lipid peroxidation indicates their involvement as pathogenic factors associated with several chronic degenerative diseases. Proteomics and mass spectrometry (MS) in the last decade have played a fundamental role in this context, allowing the demonstration of the formation of RCS-protein adducts in vitro and in vivo under different experimental conditions. In conjunction with functional and computational studies, MS has been widely applied in vitro to study the stoichiometry of the protein-RCS adduct formation, and, by identifying the site(s) of modification, to elucidate the molecular mechanisms of protein carbonylation and the physiologic impact of such modification on protein function. This review will provide an update of the MS methods commonly used in detecting and characterizing protein modification by RCS generated by lipid peroxidation, among which 4-hydroxy-trans-2-nonenal and acrolein represent the most studied and cytotoxic compounds. Research in this field, employing state-of-the-art MS, is rapidly and continuously evolving, owing also to the development of suitable derivatization and enrichment procedures enabling the improve MS detectability of RCS-protein adducts in complex biological matrices. By considering the emerging role of RCS in several human diseases, unequivocal analytical approaches by MS are needed to provide levels of intermediate diagnostic biomarkers for human diseases. This review focuses also on the different MS-based approaches so far developed for RCS-protein adduct quantification. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Reactivity, Selectivity, and Reaction Mechanisms of Aminoguanidine, Hydralazine, Pyridoxamine, and Carnosine as Sequestering Agents of Reactive Carbonyl Species: A Comparative Study.

Reactive carbonyl species (RCS) are endogenous or exogenous byproducts involved in the pathogenic mechanisms of different oxidative‐based disorders. Detoxification of RCS by carbonyl quenchers is a promising therapeutic strategy. Among the most studied quenchers are aminoguanidine, hydralazine, pyridoxamine, and carnosine; their quenching activity towards four RCS (4‐hydroxy‐trans‐2‐nonenal, methylglyoxal, glyoxal, and malondialdehyde) was herein analyzed and compared. Their ability to prevent protein carbonylation was evaluated in vitro by using an innovative method based on high‐resolution mass spectrometry (HRMS). The reactivity of the compounds was RCS dependent: carnosine efficiently quenched 4‐hydroxy‐trans‐2‐nonenal, pyridoxamine was particularly active towards malondialdehyde, aminoguanidine was active towards methylglyoxal and glyoxal, and hydralazine efficiently quenched all RCS. Reaction products were generated in vitro and were characterized by HRMS. Molecular modeling studies revealed that the reactivity was controlled by specific stereoelectronic parameters that could be used for the rational design of improved carbonyl quenchers.

An in depth proteomic analysis based on ProteoMiner, affinity chromatography and nano-HPLC–MS/MS to explain the potential health benefits of bovine colostrum

Bovine colostrum (BC), the initial milk secreted by the mammary gland immediately after parturition, is widely used for several health applications. We here propose an off-target method based on proteomic analysis to explain at molecular level the potential health benefits of BC. The method is based on the set-up of an exhaustive protein data bank of bovine colostrum, including the minor protein components, followed by a bioinformatic functional analysis. The proteomic approach based on ProteoMiner technology combined to a highly selective affinity chromatography approach for the immunoglobulins depletion, identified 1786 proteins (medium confidence; 634 when setting high confidence), which were then clustered on the basis of their biological function. Protein networks were then created on the basis of the biological functions or health claims as input. A set of 93 proteins involved in the wound healing process was identified. Such an approach also permits the exploration of novel biological functions of BC by searching in the database the presence of proteins characterized by innovative functions. In conclusion an advanced approach based on an in depth proteomic analysis is reported which permits an explanation of the wound healing effect of bovine colostrum at molecular level and allows the search of novel potential beneficial effects.

A novel high resolution MS approach for the screening of 4-hydroxy-trans-2-nonenal sequestering agents

An in vitro high resolution mass spectrometry (MS) method was set-up to test the ability of compounds, mixtures and extracts to inhibit protein carbonylation induced by reactive carbonyl species (RCS). The method consists of incubating the protein target (ubiquitin) with 4-hydroxy-trans-2-nonenal (HNE) in the presence and absence of the tested compound. After 24h of incubation, the reaction is stopped and the protein is analyzed by high-resolution MS. The extent of protein carbonylation is determined by measuring the area of the +11 multicharged peak of the HNE adduct in respect to the native form. The method was validated by measuring the effect of well-known RCS sequestering agents, namely aminoguanidine, pyridoxamine, hydralazine and carnosine, yielding a good reproducibility and the possibility to be automatable. All the compounds were found to dose-dependently inhibit the protein carbonylation with the following order of potency carnosine≈hydralazine≫aminoguanidine>pyridoxamine, as determined by calculating the UC50 values, that is the concentration required to inhibit ubiquitin carbonylation by 50%. A good correlation was found with the results obtained by measuring HNE consumption using an HPLC method optimized by a mobile phase set at pH 7.4, in order to stabilize the eluted adducts. The MS approach was then applied to test the effect of two selected natural extracts on protein carbonylation, i.e. green coffee bean extract and procyanidins from Vitis vinifera. In summary, this paper reports a validated and highly reproducible MS method to test the ability of pure compounds as well as natural extracts to act as protein carbonylation inhibitors.

Computational approaches in the rational design of improved carbonyl quenchers: focus on histidine containing dipeptides

AIM The inhibition of protein carbonylation can play therapeutic roles in several oxidative-based diseases and direct carbonyl quenching appears the most effective inhibition strategies. l-carnosine derivatives are effective and selective quenchers toward 4-hydroxy-2-nonenal even though their activity was never investigated in a fully comparable way. RESULTS The reported results revealed that anserine, homocarnosine and carnosinamide retain a remarkable quenching activity combined with a satisfactory selectivity. In silico analyses confirmed the key role of flexibility, lipophilicity and nucleophilicity parameters in rationalizing the measured reactivity. CONCLUSION This study confirms that in silico approaches can be successfully used in the rational design of improved carbonyl quenchers. Physicochemical and stereoelectronic descriptors appear really informative especially when explored by their corresponding property spaces.

Fat-Soluble Bioactive Components in Colored Rice Varieties

Bioactive components in rice vary depending on the variety and growing condition. Fat-soluble components such as γ-oryzanol, tocopherols, tocotrienols, carotenoids, and fatty acids were analyzed in brown, sugary brown, red, and black rice varieties using established high-performance liquid chromatography (HPLC) and GC methodologies. In addition, these colored rice varieties were further analyzed using a high-resolution liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) (LTQ-Orbitrap XL) to identify the [M-H](-) ions of γ-oryzanol, ranging from m/z 573.3949 to 617.4211. The highest content of tocopherols (α-, 1.5; γ-, 0.5 mg/100 g) and carotenoids (lutein 244; trans-β carotene 25 μg/100 g) were observed in black rice; tocotrienols (α-, 0.07; γ-, 0.14 mg/100 g) in red rice, and γ-oryzanol (115 mg/100 g) in sugary brown rice. In all colored rice varieties, the major fatty acids were palmitic (16:0), oleic (18:1n-9), and linoleic (18:2n-6) acids. When the γ-oryzanol components were further analyzed by LC-MS/MS, 3, 10, 8, and 8 triterpene alcohols or sterol ferulates were identified in brown, sugary brown, red, and black rice varieties, respectively. Such structural identification can lead to the elucidation of biological function of each component at the molecular level. Consumption of colored rice rich in beneficial bioactive compounds may be a useful dietary strategy for achieving optimal health.

A method to produce fully characterized ubiquitin covalently modified by 4-hydroxy-nonenal, glyoxal, methylglyoxal, and malondialdehyde

ABSTRACT Reactive carbonyl species (RCS) and the corresponding protein adducts (advanced glycoxidation or lipoxidation end products, i.e. AGEs and ALEs) are now widely studied from different points of view, since they can be considered as biomarkers, pathogenic factors, toxic mediators and drug targets. One of the main limits of the research in this field is the lack of standardized and fully characterized AGEs and ALEs to be used for biological, toxicological, and analytical studies. In this work, we set up a procedure to prepare and fully characterize a set of AGEs and ALEs by incubating ubiquitin – a model protein selected as target for carbonylation – with four different RCS: 4-hydroxy-trans-2-nonenal (HNE), methylglyoxal (MGO), glyoxal (GO), and malondialdehyde (MDA). After 24 h of incubation, the extent of protein carbonylation was estimated using a recently developed quantitative strategy based on high-resolution mass spectrometry. The resulting AGEs and ALEs were fully characterized by both intact protein and bottom-up analyses in terms of: stoichiometry of the total amount of modified protein, elucidation of the structure of the RCS-deriving adducts, and localization of the RCS-modified amino acids. Each RCS exhibited different reactivity toward ubiquitin, as detected by quantifying the extent of protein modification. The order of reactivity was MGO > GO > HNE > MDA. A variety of reaction products was identified and mapped on lysine, arginine, and histidine residues of the protein. In summary, a highly standardized and reproducible method to prepare fully characterized AGEs/ALEs is here presented.

The secrets of Oriental panacea: Panax ginseng.

UNLABELLED The Panax ginseng root proteome has been investigated via capture with combinatorial peptide ligand libraries (CPLL) at three different pH values. Proteomic characterization by SDS-PAGE and nLC–MS/MS analysis, via LTQ-Orbitrap XL, led to the identification of a total of 207 expressed proteins. This quite large number of identifications was achieved by consulting two different plant databases: P. ginseng and Arabidopsis thaliana. The major groups of identified proteins were associated to structural species (19.2%), oxidoreductase (19.5%), dehydrogenases (7.6%) and synthases (9.0%). For the first time, an exploration of protein–protein interactions was performed by merging all recognized proteins and building an interactomic map, characterized by 196 nodes and 1554 interactions. Finally a peptidomic analysis was developed combining different in-silico enzymatic digestions to simulate the human gastrointestinal process: from 661 generated peptides, 95 were identified as possible bioactives and in particular 6 of them were characterized by antimicrobial activity. The present report offers new insight for future investigations focused on elucidation of biological properties of P. ginseng proteome and peptidome. BIOLOGICAL SIGNIFICANCE Ginseng is a traditional oriental herbal remedy whose use is very diffused in all the world for its numerous pharmacological effects. However, the exact mechanism of action of ginseng components, both ginsenosides and proteins, is still unidentified. So the common use of ginseng requires strict investigations to assess both its efficiency and its safety. Although many reports have been published regarding the pharmacological effects of ginseng, little is known about the biochemical pathways of root. Proteomics analysis could be useful to elucidate the physiological pathways. In this manuscript, an integrated approach to proteomics and peptidomics will usher in exploration of Panax ginseng proteins and proteolytic peptides, obtained by in-silico gastrointestinal digestion, characterized by antimicrobial action. The present research would pave the way for better knowledge of metabolic functions connected with ginseng proteome and provide with new information necessary to understand better antimicrobial activity of P. ginseng.

Advanced glycation end products of beta2-microglobulin in uremic patients as determined by high resolution mass spectrometry.

By using a high resolution top-down and bottom-up approach we identified and characterized the AGEs of beta2-microglobulin (β2-m) formed by incubating the protein in the presence of glucose and of the main reactive carbonyl species. Glucose induced glycation on the N-terminal residue, while glyoxal (GO) and methylglyoxal (MGO) covalently reacted with Arg3. Carboxymethyl (CM-R) and imidazolinone (R-GO) derivatives were identified in the case of GO and carboxyethyl arginine (CE-R) and methyl-imidazolinone (R-MGO) for MGO. Interestingly, α,β-unsaturated aldehydes [4-hydroxy-2-nonenal (HNE); 4-oxo-2-nonenal (ONE); acrolein (ACR)] did not induce any covalent modifications up to 100μM. The different reactivity of β2-m towards the different RCS was then rationalized by molecular modeling studies. The MS method was then applied to fully characterize the AGEs of β2-m isolated from the urine of uremic subjects. CM-R, CE-R and R-MGO were easily identified on Arg3 and their relative abundance in respect to the native protein determined by a semi-quantitative approach. Overall, the AGEs content of urinary β2-m ranged from 0.2 to 1% in uremic subjects. The results here reported offer novel insights and technical achievements for a potential biological role of AGEs-β2-m in pathological conditions.

CHAPTER 8:Carnosine and Derivatives as Inhibitors of Protein Covalent Modifications Induced by Reactive Carbonyl Species

Reactive carbonyl species (RCS) are a class of reactive compounds that covalently react with nucleophilic substrates such as proteins. Reaction of RCS with proteins is involved in the onset and propagation of several oxidative-based diseases including atherosclerosis, diabetes and neurological disorders. RCS are efficiently detoxified by phase I and II metabolic pathways. More recently it has been found that histidine dipeptides, including carnosine and anserine, are involved in RCS detoxification by forming unreactive covalent adducts. Such a detoxification reaction was first demonstrated in vitro and then in vivo, in both rodents and humans, by detecting the conjugated metabolites between carnosine and the most reactive and abundant RCS, such as acrolein and hydroxynonenal. Furthermore, in animal models of metabolic syndrome and atherosclerosis, carnosine treatment induced a significant protective effect, which was associated with a reduction of RCS-modified proteins (ALEs and AGEs) and with the excretion of carnosine-RCS adducts. Such evidence, confirmed by different independent research groups, indicates that in physiopathological conditions characterized by oxidative damage, carnosine can act as an efficient RCS detoxification agent, reducing protein RCS modification and leading to a beneficial effect. Such studies prompted the discovery of carnosine derivatives resistant to carnosinase as a novel class of bioactive compounds.