Mass spectrometry‐based proteomics of oxidative stress: Identification of 4‐hydroxy‐2‐nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins

Modification of proteins by 4‐hydroxy‐2‐nonenal (HNE), a reactive by‐product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age‐related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff‐base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff‐base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS “signatures” of HNE‐modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE‐modified lysozyme into an electrospray quadrupole time‐of‐flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC‐MS/MS, we found that, in addition to N‐terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.     

Proximity-induced amino-yne reaction for selective MDM4 conjugation via propargylated sulfonium

Whilst most bioorthogonal reactions focus on targeting binding-site cysteine residues, proximity-induced reactivity effect ensures that reaction also occurs at nucleophilic lysine residues. We report one example here that the propargylated-sulfonium center undergoes a nucleophilic reaction with lysine residue via proximity-induced conjugation. This propargylated-sulfonium tethered peptide resulting from a facile propargylation of thiolethers, enables amino-yne reaction at the selected lysine on MDM4 protein. This strategy represents a viable approach of lysine-targeted covalent inhibition in proximity.     

Halomethyl-Triazoles for Rapid,Site-Selective Protein Modification

Post-translational modifications (PTMs) are used by organisms to control protein structure and function after protein translation, but their study is complicated and their roles are not often well understood as PTMs are difficult to introduce onto proteins selectively. Designing reagents that are both good mimics of PTMs, but also only modify select amino acid residues in proteins is challenging. Frequently, both a chemical warhead and linker are used, creating a product that is a misrepresentation of the natural modification. We have previously shown that biotin-chloromethyl-triazole is an effective reagent for cysteine modification to give S-Lys derivatives where the triazole is a good mimic of natural lysine acylation. Here, we demonstrate both how the reactivity of the alkylating reagents can be increased and how the range of triazole PTM mimics can be expanded. These new iodomethyl-triazole reagents are able to modify a cysteine residue on a histone protein with excellent selectivity in 30 min to give PTM mimics of acylated lysine side-chains. Studies on the more complicated, folded protein SCP-2L showed promising reactivity, but also suggested the halomethyl-triazoles are potent alkylators of methionine residues.     

Covalent binding of human serum albumin and ovalbumin by chloramine-T and chemical modification of the proteins

The covalent binding of ³⁵S-chloramine-T to human resum albumin (HSA) and ovalbumin is described. At pH 6.5, up to 24 chloramine-T molecules were found to be covalently bound per molecule of HSA; with ovalbumin the binding was only 5–7 molecule per protein molecule. Binding was accompanied by extensive modification of methionine, cysteine, histidine, tyrosine and lysine. Three new peaks appeared in the amino acid profiles of the modified proteins; two were identified as 1-aminoadipic acid (oxidation of lysine) and 3-chlorotyrosine. The most sites for covalent binding are lysine residues.     

Oxidation of bovine serum albumin: identification of oxidation products and structural modifications

Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long‐lived proteins. The metal‐catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system. Mass spectrometry, combined with off‐line nano‐liquid chromatography, was used to identify modifications in amino acid residues. We have found 106 different residues oxidatively damaged, being the main oxidized residues lysines, cysteines, arginines, prolines, histidines and tyrosines. Besides protein hydroxyl derivatives and oxygen additions, we detected other modifications such as deamidations, carbamylations and specific amino acid oxidative modifications. The oxidative damage preferentially affects particular subdomains of the protein at different time‐points. Results suggest the oxidative damage occurs first in exposed regions near cysteine disulfide bridges with residues like methionine, tryptophan, lysine, arginine, tyrosine and proline appearing as oxidatively modified. The damage extended afterwards with further oxidation of cysteine residues involved in disulfide bridges and other residues like histidine, phenylalanine and aspartic acid. The time‐course evaluation also shows the number of oxidized residues does not increase linearly, suggesting that oxidative unfolding of albumin occurs through a step‐ladder mechanism. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative display of the redox status of proteins with maleimide‐polyethylene glycol tagging

Cysteine oxidation, either biologically reversible or irreversible, is the main posttranslational modification associated with redox signaling and oxidative stress. Maleimide‐polyethylene glycol (m‐PEG) has been used to detect reversibly oxidized proteins by reacting to the reduced cysteine residues leading to mobility shift in immunoblots; a method called PEG‐switch. With PEG‐switch, both reduced and oxidized proteins can be observed on the same immunoblot simultaneously, providing a simple quantitative measurement for protein thiol modifications. In this report, we optimized the assay conditions and exploited the applications of PEG‐switch in quantitation of the extent of protein thiol oxidation in cells in response to H₂O₂ and insulin. In addition, we have proposed a redox scoring system for measuring the redox status of any given protein from the m‐PEG immunoblot. Our results provided quantitative data showing that two cysteine residues of protein tyrosine phosphatase 1B are prone to oxidation following insulin treatment in cultured HeLa cells.     

Cross-mixing study of a poisonous Cestrum species,Cestrum diurnum in herbal raw material by chemical fingerprinting using LC-ESI-QTOF-MS/MS

Poisonous plants are widely distributed and may have risk of phytotoxicity upon mixing with medicinal plants. Several species of Cestrum genus are poisonous and linked with many serious health issues. In the present study, cross-mixing of a toxic plant, Cestrum diurnum with morphologically resembling medicinal plant, Adhatoda vasica was studied using chemical fingerprinting approach. LC-ESI-MS/MS tool was used to develop the chemical fingerprints of three toxic species of Cestrum, including, C. diurnum, C. nocturnum and C. parqui. Total forty-three compounds were identified using high-resolution LC-ESI-MS/MS data comparison. Chemometric analyses were done to compare the distribution of identified compounds present in these Cestrum species. One of the identified compounds, nornicotine (a toxic compound) was also quantified using LC-IT-MS/MS. Adulteration study was conducted by mixing toxic C. diurnum in A. vasica with various ratios (w/w) and five differentiable compounds were identified to detect the adulteration. The method was able to detect up to the limit of 5% mixing of toxic C. diurnum. Moreover, cytotoxicity of the methanolic extracts of these three species were also studied on normal human PBMC (peripheral blood mononuclear cells) and all found to be toxic, while the C. nocturnum showed the highest level of toxicity with the IC₅₀ 12.5 μg/mL.     

Possible Evidence of Amide Bond Formation Between Sinapinic Acid and Lysine-Containing Bacterial Proteins by Matrix-Assisted Laser Desorption/Ionization (MALDI) at 355?nm

We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, Hde, and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight tandem mass spectrometry (TOF-TOF-MS/MS) and post-source decay (PSD). We also reported the absence of adduct formation when using ??-cyano-4-hydroxycinnamic acid (CHCA) matrix. Further mass spectrometric analysis of disulfide-intact and disulfide-reduced over-expressed HdeA and HdeB proteins from lysates of gene-inserted E. coli plasmids suggests covalent attachment of SA occurs not at cysteine residues but at lysine residues. In this revised hypothesis, the attachment of SA is preceded by formation of a solid phase ammonium carboxylate salt between SA and accessible lysine residues of the protein during sample preparation under acidic conditions. Laser irradiation at 355?nm of the dried sample spot results in equilibrium retrogradation followed by nucleophilic attack by the amine group of lysine at the carbonyl group of SA and subsequent amide bond formation and loss of water. The absence of CHCA adducts suggests that the electron-withdrawing effect of the ??-cyano group of this matrix may inhibit salt formation and/or amide bond formation. This revised hypothesis is supported by dissociative loss of SA (?224?Da) and the amide-bound SA (?206?Da) from SA-adducted HdeA and HdeB ions by MS/MS (PSD). It is proposed that cleavage of the amide-bound SA from the lysine side-chain occurs via rearrangement involving a pentacyclic transition state followed by hydrogen abstraction/migration and loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal (?206?Da).     

A Stable Isotopic Pre-digestion Labeling Method for Protein Quantitative Analysis Using Matrix-assisted Laser Desorption/ionization Mass Spectrometry

As an extension of our previous work, here a strategy was demonstrated for protein identification and quantification analyses utilizing a combination of stable isotope chemical labeling with subsequent denaturation, enzymatic digestion and matrix assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Using [d₀]‐ and [d₆]‐4,6‐dimethoxy‐2‐(methylsulfonyl)pyrimidine ([d₀]‐/[d₆]‐DMMSP), stable isotopic labels were incorporated before digestion. The comparative samples were combined before labeling after digestion, thus biases resulting from differences in sample digestion were avoided and the higher accuracy of quantification could be attained. The labeling was spatial‐selective to particular residues of cysteine, lysine, and tyrosine before denaturation, which could lead to a better universality of the strategy for cysteine‐free proteins. In addition, some lysine residues were blocked after labeling, the partly destroyed recognition sites could simplify the trypsin hydrolysates and hence facilitate the MS complexity. Together, our one‐step labeling strategy combined several desirable properties such as spatial‐selective labeling, reliability of quantitative results, simplification of analysis of complex systems and direct analysis with minimum sample handling. Our results demonstrate the usefulness of the method for analyzing lysozyme in egg white. The method was expected to provide a new powerful tool for comparative proteome research.     

Covalent attachment and dissociative loss of sinapinic acid to/from cysteine-containing proteins from bacterial cell lysates analyzed by MALDI-TOF-TOF mass spectrometry

We report covalent attachment via a thiol ester linkage of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA) to cysteine-containing protein biomarkers from bacterial cell lysates of E. coli analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry when using SA as the matrix. Evidence to support this conclusion is the appearance of additional peaks in the MS spectra when using SA, which are absent when using α-cyano-4-hydroxycinnamic acid (HCCA). The additional peaks appear at a mass-to-charge (m/z) ∼208 greater to the m/z of a more abundant protein ion peak. Protein biomarkers were identified by tandem mass spectrometry (MS/MS) using a MALDI time-of-flight/time-of-flight (TOF-TOF) mass spectrometer and top-down proteomics. Three protein biomarkers, HdeA, HdeB, and homeobox or YbgS (each containing two cysteine residues) were identified as having reactivity to SA. Non-cysteine-containing protein biomarkers showed no evidence of reactivity to SA. MS ions and MS/MS fragment ions were consistent with covalent attachment of SA via a thiol ester linkage to the side-chain of cysteine residues. MS/MS of a protein biomarker ion with a covalently attached SA revealed fragment ion peaks suggesting dissociative loss SA. We propose dissociative loss of SA is facilitated by a pentacyclic transition-state followed by proton abstraction of the β-hydrogen of the bound SA by a sulfur lone pair followed by dissociative loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal. The apparent reactivity of SA to cysteine/disulfide-containing proteins may complicate identification of such proteins, however the apparent differential reactivity of SA and HCCA toward cysteine/disulfide-containing proteins may be exploited for identification of unknown cysteine-containing proteins.     

The Proteome‐Wide Potential for Reversible Covalency at Cysteine

Reversible covalency, achieved with, for instance, highly electron‐deficient olefins, offers a compelling strategy to design chemical probes and drugs that benefit from the sustained target engagement afforded by irreversible compounds, while avoiding permanent protein modification. Reversible covalency has mainly been evaluated for cysteine residues in individual kinases and the broader potential for this strategy to engage cysteines across the proteome remains unexplored. Herein, we describe a mass‐spectrometry‐based platform that integrates gel filtration with activity‐based protein profiling to assess cysteine residues across the human proteome for both irreversible and reversible interactions with small‐molecule electrophiles. Using this method, we identify numerous cysteine residues from diverse protein classes that are reversibly engaged by cyanoacrylamide fragment electrophiles, revealing the broad potential for reversible covalency as a strategy for chemical‐probe discovery.     

‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

Protein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. However, a common problem associated with the use of selective chemical labeling methods for mapping protein solvent accessible residues is that when a complicated peptide mixture resulting from a large protein or protein complex is analyzed, the modified peptides may be difficult to identify and characterize amongst the largely unmodified peptide population (i.e., the ‘needle in a haystack’ problem). To address this challenge, we describe here the development of a strategy involving the synthesis and application of a novel ‘fixed charge’ sulfonium ion containing lysine-specific protein modification reagent, S,S′-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), coupled with capillary HPLC-ESI-MS, automated CID-MS/MS, and data-dependant neutral loss mode MS³ in an ion trap mass spectrometer, to map the surface accessible lysine residues in a small model protein, cellular retinoic acid binding protein II (CRABP II). After reaction with different reagent:protein ratios and digestion with Glu-C, modified peptides are selectively identified and the number of modifications within each peptide are determined by CID-MS/MS, via the exclusive neutral loss(es) of dimethylsulfide, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility) of the peptide. The observation of these characteristic neutral losses are then used to automatically ‘trigger’ the acquisition of an MS³ spectrum to allow the peptide sequence and the site(s) of modification to be characterized. Using this approach, the experimentally determined relative solvent accessibilities of the lysine residues were found to show good agreement with the known solution structure of CRABP II.     

Selective tryptophan modification with rhodium carbenoids in aqueous solution

A new transition metal-based reaction has been developed for the selective modification of tryptophan residues on protein substrates. After activation of vinyl-substituted diazo compounds by Rh2(OAc)4, the resulting metallocarbenoid intermediates were found to modify indoles in aqueous media despite competing reactions with water. Both N- and 2-substituted indole products were observed in the reaction. Following initial small-molecule studies, the reaction was performed on two protein substrates. Both myoglobin and subtilisin Carlsberg were modified readily in aqueous solution, and the tryptophan selectivity of the reactions was confirmed through MS analyses of trypsin digest fragments. It was also demonstrated that myoblobin concentrations as low as 10 muM still led to appreciable levels of modification. Reconstitution experiments confirmed that myoglobin retained its ability to bind heme following modification.     

On-line electrochemical tagging of cysteines in proteins during nanospray

An electrochemical modification of free cysteine residues is studied and characterized by means of quinone addition. Taking advantage of the electrolytic nature of electrospray interfaces (ESI), an electrochemical tagging is performed prior to mass spectrometry (MS) analyses. The tagging has been studied by MS and different mechanisms, involving electrochemical and/or chemical steps, could be characterized. It is demonstrated that the present nanospray is a very efficient tool to obtain cysteine modification. Using the high voltage electrode of the nanospray interface to perform protein specific tagging is a novel method that can be associated to analytical or preparative techniques, such as digestion of proteins or capillary electrophoresis, for post-column modifications.     

A cationic cysteine-hydrazide as an enrichment tool for the mass spectrometric characterization of bacterial free oligosaccharides

In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This hydrolysis results in the same molecular structure as the oligosaccharide that is transferred to a protein to be glycosylated. This allowed for the general elucidation of the fOS-branched structures and monosaccharides from a number of species using standard enrichment and mass spectrometry methods. To aid characterization of fOS, hydrazide chemistry has often been used for chemical modification of the reducing part of oligosaccharides resulting in better selectivity and sensitivity in mass spectrometry; however, the removal of the unreacted reagents used for the modification often causes the loss of the sample. Here, we develop a more robust method for fOS purification and characterize glycostructures using complementary tandem mass spectrometry (MS/MS) analysis. A cationic cysteine hydrazide derivative was synthesized to selectively isolate fOS from periplasmic fractions of bacteria. The cysteine hydrazide nicotinamide (Cyhn) probe possesses both thiol and cationic moieties. The former enables reversible conjugation to a thiol-activated solid support, while the latter improves the ionization signal during MS analysis. This enrichment was validated on the well-studied Campylobacter jejuni by identifying fOS from the periplasmic extracts. Using complementary MS/MS analysis, we approximated data of a known structure of the fOS from Campylobacter concisus. This versatile enrichment technique allows for the exploration of a diversity of protein glycosylation pathways.     

Concise synthesis of multiply substituted stereodefined cis,cis-2,4-diene-1,6-dials, cis,cis-2,4-diene-1,6-diols and further applications

Reactions of 1,4-dilithio-1,3-dienes with DMF afforded multiply substituted stereodefined cis,cis-2,4-diene-1,6-dials in high yields. Treatment of these 1,6-dials with LiAlH₄ or RLi resulted in the formation of their corresponding 1,6-diols. These bifunctional compounds, cis,cis-2,4-diene-1,6-dials and -1,6-diols are otherwise not readily available. Further reaction of these 1,6-diols with an aldehyde catalyzed by strong acids led to the formation of oxacycles of novel structures.     

Late-stage modification of peptides and proteins at cysteine with diaryliodonium salts

The modification of peptides and proteins has emerged as a powerful means to efficiently prepare high value bioconjugates for a range of applications in chemical biology and for the development of next-generation therapeutics. Herein, we report a novel method for the chemoselective late-stage modification of peptides and proteins at cysteine in aqueous buffer with suitably functionalised diaryliodonium salts, furnishing stable thioether-linked synthetic conjugates. The power of this new platform is showcased through the late-stage modification of the affibody zEGFR and the histone protein H2A.

New operationally simple platform for the chemoselective arylation of cysteine in peptides and proteins to access a variety of high value bioconjugates.     

Peptide and protein assays using customizable bio-affinity arrays combined with ambient ionization mass spectrometry

High-throughput identification and quantification of protein/peptide biomarkers from biofluids in a label-free manner is achieved by interfacing bio-affinity arrays (BAAs) with nano-electrospray desorption electrospray ionization mass spectrometry (nano-DESI-MS). A wide spectrum of proteins and peptides ranging from phosphopeptides to cis-diol biomolecules as well as thrombin can be rapidly extracted via arbitrarily predefined affinity interactions including coordination chemistry, covalent bonding, and biological recognition. An integrated MS platform allows continuous interrogation. Profiling and quantitation of dysregulated phosphopeptides from small-volume (∼5 μL) serum samples has been successfully demonstrated. As a front-end device adapted to any mass spectrometer, this MS platform might hold much promise in protein/peptide analysis in point-of-care (POC) diagnostics and clinical applications.

Customizable bio-affinity arrays were interfaced with ambient ionization mass spectrometry for high-throughput assays of protein/peptide biomarkers in biofluids.     

Lysine‐directed staining of proteins for MS‐based analyses

Visualization of proteins and MS‐based analyses are elemental tasks in modern biochemistry. Nevertheless, reports about covalent protein dyes and their suitability for subsequent MS experiments remain scarce. In a recent work, we demonstrated that covalent prestaining of proteins with Uniblue A drastically speeds up proteomic workflows. The present study introduces dabsyl chloride as another truly MS‐compatible protein stain. Remarkably, although Uniblue A and dabsyl chloride employ different nucleophilic reaction mechanisms, both are highly specific for lysine residues. The predictable peptide modifications allow easy integration into state‐of‐the‐art bioinformatic workflows. Further, lysine‐directed derivatizations with hydrophobic reagents such as dabsyl chloride complement the cysteine‐directed ALiPHAT strategy for increasing the sensitivity of peptide identifications.     

GC/MS and ESI/MS identification of the new generation plasticizers - cis and trans isomers of some 1,2-cyclohexane dicarboxylic acid di(n- and isononyl) esters

1,2-Cyclohexane dicarboxylic acid diisononyl ester is also known as diisononyl cyclohexane-1,2-dicarboxylate (DINCH) is a complex mixture of the hydrogenation products of diisononyl benzene-1,2-dicarboxylate (DINP). They are the new generation plasticizers used instead of the dialkyl benzene-1,2-dicarboxylate in order to improve the flexibility of polymers (mainly PVC) and strongly reduce the toxic effects on human health during their release from polymers to the environment. The identification of these compounds was done by syntheses of some DINCH constituents, i.e. cis and trans isomers of three di(n- and isononyl) cyclohexane-1,2-dicarboxylates, such as di(3,5,5-trimethylhexyl) cyclohexane-1,2-dicarboxylates, di(2-methyloctyl) cyclohexane-1,2-dicarboxylates and dinonyl cyclohexane-1,2-dicarboxylates by catalytic hydrogenation of appropriate DINP and their analyses by gas chromatography – mass spectrometry (GC/MS) and electrospray - mass spectrometry (ESI/MS) methods. Both GC data (values of the retention times t_R and arithmetic retention indices I_A) and mass spectra obtained for these isomers allowed to determine their chemical structures. ESI/MS mode of analysis of these compounds gives the knowledge about their mass fragmentation without the differentiation between individual cis and trans isomers.