Investigation of tyrosine nitration and nitrosylation of angiotensin II and bovine serum albumin with electrospray ionization mass spectrometry

Protein tyrosine nitration is one of the important regulatory mechanisms in various cellular phenomena such as cell adhesion, endo/exo-cytosis of cellular materials, and signal transduction. In the present study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) with a linear ion-trap mass spectrometer was applied for identification of nitrated proteins and localization of the modified tyrosine residues. When angiotensin II(DRVYIHPF) was nitrated in vitro with tetranitromethane (TNM), the mass spectrum showed a shift of +45 Da which corresponded to tyrosine nitration. An additional +29 Da mass shift was also detected by ESI-MS. This differed from nitrated peptide analysis with matrix-associated laser desorption/ionization mass spectrometry (MALDI-MS), which showed oxygen neutral loss from the nitrated tyrosine residues upon laser irradiation. Hence the +29 Da mass shift of the nitrated peptide observed by ESI-MS suggested the introduction of an NO group for nitrosylation of tyrosine residues. To confirm this in vitro nitrosylation on the protein level, bovine serum albumin was in vitro nitrated with TNM and analyzed by ESI-MS/MS. As expected, +29 as well as +45 Da mass shifts were detected, and the +29 Da mass shift was found to correspond to the modification on tyrosine residues by NO. Although the chemical mechanism by which this occurs in ESI-MS is not clear, the +29 Da mass shift could be a new potential marker of nitrosylated peptides.     

Protein-bound tyrosine oxidation,nitration and chlorination by-products assessed by ultraperformance liquid chromatography coupled to tandem mass spectrometry

Background

Free radicals cause alterations in cellular protein structure and function. Oxidized, nitrated, and chlorinated modifications of aromatic amino acids including phenylalanine and tyrosine are reliable biomarkers of oxidative stress and inflammation in clinical conditions.

Objective

To develop, validate and apply a rapid method for the quantification of known hallmarks of tyrosine oxidation, nitration and chlorination in plasma and tissue proteins providing a snapshot of the oxidative stress and inflammatory status of the organism and of target organs respectively.

Material and Methods

The extraction and clean up procedure entailed protein precipitation, followed by protein re-suspension and enzymatic digestion with pronase. An Ultra Performance Liquid Chromatography–tandem Mass Spectrometry (UPLC-MS/MS) method was developed to quantify protein released ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-nitrotyrosine (3NO₂-Tyr) and 3-chlorotyrosine (3Cl-Tyr) as well as native phenylalanine (Phe) and tyrosine (p-Tyr) in plasma and tissue from a validated hypoxic newborn piglet experimental model.

Results

In plasma there was a significant increase in the 3NO₂-Tyr/p-Tyr ratio. On the other hand m-Tyr/Phe and 3Cl-Tyr/p-Tyr ratios were significantly increased in liver of hypoxic compared with normoxic animals. Although no significant differences were found in brain tissue, a clear tendency to increased ratios was observed under hypoxic conditions.

Conclusions

UPLC-MS/MS has proven suitable for the analysis of plasma and tissue samples from newborn piglets. The analysis of biomarkers of protein oxidation, nitration and chlorination will be applied in future studies aiming to provide a deeper insight into the mechanisms of oxidation-derived protein modification caused during neonatal asphyxia and resuscitation.     

Characterization of proteins by mass spectrometry. Invited lecture.

Plasma desorption and fast atom bombardment mass spectrometry have in the last decade demonstrated the potential of mass spectrometry for protein studies. The recently developed matrix-assisted laser desorption and electrospray mass spectrometry have expanded the analytical potential of mass spectrometry to cover nearly all proteins. The type of information obtained with the four methods is described and their performances are compared. The potential of combining mass spectrometric relative molecular mass information on proteins with the information contained in protein sequence databases is outlined and some typical fields of application of mass spectrometry in protein chemistry are described. The need for the full integration of mass spectrometry in the protein laboratory is discussed.     

Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.

Hydrogen-exchange electrospray-ionization mass spectrometry is demonstrated to be an effective new method for probing conformational changes of proteins in solutions. The method is based on the mass spectrometric measurement of the extent of hydrogen/deuterium exchange that occurs in different protein conformers over defined periods of time. Results are presented in which hydrogen-exchange electrospray-ionization mass spectrometry is used to probe conformational changes in bovine ubiquitin induced by the addition of methanol to aqueous acidic solutions of the protein.     

Identification of the tyrosine nitration sites in human endothelial nitric oxide synthase by liquid chromatography-mass spectrometry

The formation of nitric oxide (NO) in biological systems has led to the discovery of a number of post- translational protein modifications that can affect biological conditions such as vasodilation. Studies both from our laboratory and others have shown that beside its effect on cGMP generation from soluble guanylate cylcase, NO can produce protein modifications through both S-nitrosylation of cysteine residues. Previously, we have identified the potential S-nitrosylation sites on endothelial NO synthase (eNOS). Thus, the goal of this study was to further increase our understanding of reactive nitrogen protein modifications of eNOS by identifing tyrosine residues within eNOS that are susceptible to nitration in vitro. To accomplish this, nitration was carried out using tetranitromethane followed by tryptic digest of the protein. The resulting tryptic peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) and the position of nitrated tyrosines in eNOS were identified. The eNOS sequence contains 30 tyrosine residues and our data indicate that multiple tyrosine residues are capable of being nitrated. We could identify 25 of the 30 residues in our tryptic digests and 19 of these were susceptible to nitration. Interstingly, our data identified four tyrosine residues that can be modified by nitration that are located in the region of eNOS responsible for the binding to heat shock protein 90 (Hsp90), which is responsible for ensuring efficient coupling of eNOS.     

Sequencing membrane proteins by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) is an attractive technique for sequencing membrane proteins because it can be applied to peptides in mixtures that are difficult to separate chromatographically. To evaluate the suitability of MS/MS sequencing for membrane proteins and to develop protocols for the preparation of the cleaved peptides, we employed the well characterized apoproteins of bacteriorhodopsin and bovine rhodopsin, i.e. bacterioopsin and opsin, respectively. Without separation, nine out of ten peptides resulting from cyanogen bromide cleavage of bacterioopsin were detected by fast atom bombardment MS, the single undetected fragment being a tetrapeptide that was presumably hidden in the low-m/z matrix background. Furthermore, MS/MS was used to confirm the sequence of all the peptides detected with m/z values below 3.5 kDa (40% of the protein). Bovine opsin was analyzed in a similar fashion. Tandem MS/MS has thus allowed the sequencing of substantial portions of two integral membrane proteins by the analysis of unseparated peptide mixtures, demonstrating for the first time that this technique can obviate some of the most serious difficulties associated with sequencing membrane proteins, namely the difficult-to-achieve separation of the ‘sticky’ peptide fragments.     

Fingerprinting proteins coupled with polymers by mass spectrometry: Investigation of polyethylene glycol-conjugated superoxide dismutase

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry was investigated as a method for the rapid determination of the extent of polymer coupling in polyethylene glycol- (PEG) conjugated superoxide dismutase (SOD). PEG-conjugated SOD, an antioxidant with an extended in vivo circulation lifetime compared to that of superoxide dismutase, is being evaluated as an effective therapeutic agent for the treatment of injuries and arthritis. The mass spectra of a standard batch of PEG-conjugated bovine SOD showed the presence of identifiable and well resolved peaks that correspond to 0–7 PEG molecules attached to bovine SOD. The area of each of the peaks provides a determination of the amount of PEG-conjugated SOD with a given number of bound PEG groups. SOD is a noncovalent dimer of two identical subunits that dissociates in MALDI. The information obtained in the mass spectra thus corresponds to a monomer of SOD. Each SOD monomer contains 10 lysines, which are the sites of PEG-conjugation. Multiple MALDI determinations of two batches of samples indicated good reproducibility for routine determination of the extent of polymer content. The amount of PEG-conjugated SOD that contained a given number of PEG molecules, determined by MALDI, was compared with the value deduced from the amount of PEG-conjugation at each attachment site measured by a peptide mapping method. Agreement between the data obtained in the two techniques (MALDI and peptide mapping) indicates that MALDI may be used to obtain quantitative information on PEG-conjugated SOD to determine the amounts of PEG-conjugated protein each with a different number of PEG groups attached. Measurement of several batches of samples stored at a higher temperature showed a lower extent of PEG-conjugation in PEG-conjugated SOD. This reduction in the PEG content resulted from the PEG-deconjugation of PEG-conjugated SOD at a higher temperature. Thus, MALDI can be used to examine the stability of PEG-conjugated SOD. The high sensitivity, relatively straightforward data interpretation, speed of analyses, and good reproducibility in measurements make this technique a useful analytical tool for fingerprinting PEG-conjugated SOD as well as potentially other polymer-conjugated proteins.     

Conversion of 3‐nitrotyrosine to 3‐aminotyrosine residues facilitates mapping of tyrosine nitration in proteins by electrospray ionization–tandem mass spectrometry using electron capture dissociation

Protein tyrosine nitration is associated with oxidative stress and various human diseases. Tandem mass spectrometry has been the method of choice for the identification and localization of this posttranslational modification to understand the underlying mechanisms and functional consequences. Due to the electron predator effect of the nitro group limiting fragmentation of the peptide backbone, electron‐based dissociation has not been applicable, however, to nitrotyrosine‐containing peptides. A straightforward conversion of the nitrotyrosine to the aminotyrosine residues is introduced to address this limitation. When tested with nitrated ubiquitin and human serum albumin as model proteins in top‐down and bottom‐up approaches, respectively, this chemical derivatization enhanced backbone fragmentation of the corresponding nitroproteins and nitropeptides by electron capture dissociation (ECD). Increased sequence coverage has been obtained by combining in the bottom‐up strategy the conversion of nitrotyrosine to aminotyrosine and introducing, in addition to trypsin, a further digesting enzyme of complementary specificity, when protein nitration was mapped by liquid chromatography–electrospray ionization tandem mass spectrometry using both collision‐induced dissociation (CID) and ECD. Copyright © 2012 John Wiley & Sons, Ltd.     

Investigation of alkaloid N-oxides by secondary-ion mass spectrometry

An advantage of theSIMS method for investigating alkaloid N-oxides has been shown. In the case of N-oxides of steroid, diterpene, tropane, and pyrrolizidine alkaloids, fragments including the oxygen of the N-oxide group are revealed. The protonation of N-oxide molecules in a glycerol matrix takes place mainly through the negatively polarized oxygen atom of the N-oxide function. On fragmentation, theMH ⁺ ions of N-oxides of monoester tropane and pyrrolizidine alkaloids tend to form fragmentary ions of the protonated forms of the aminoalcohols (A+). The energies of the metastable transitionsMH ⁺ A⁺ have been calculated for the Noxides of the pyrrolizidine alkaloids viridif lorine, trachelanthamine, and echinatine.Institute of the Chemistry of Plant Substances [IKhRV], Academy of Sciences of the Republic of Uzbekistan [AN RUz], Tashkent, fax (3712) 89 14 75. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 833–837, November-December, 1995. Original article submitted February 20, 1995.     

Determination of phenylalanine and tyrosine by liquid chromatography/mass spectrometry

Phenylketonuria is a common metabolic disorder disease. Those affected appear normal at birth, but without treatment they develop severe psychomotor retardation. Throughout life, they must control their blood levels of phenylalanine (Phe) and consume a diet containing adequate amounts of Phe and tyrosine (Tyr). We have developed a liquid chromatographic/mass spectrometric (LC/MS) method for the quantitative evaluation of Phe and Tyr in food samples. This method takes advantage of the good separation of LC and the selective and reliable quantification provided by MS in the electrospray ionization mode. The LC/MS method is very suitable for the determination of selected amino acids in various matrixes. It is sensitive to levels as low as about 0.30 ppm for Tyr and 0.70 ppm for Phe and robust. Nearly 100 nondietary food samples were analyzed by the developed method.     

Identification of gel-separated tumor marker proteins by mass spectrometry

Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these over-expressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.     

Thermal unfolding of proteins probed by laser spray mass spectrometry

The stability and conformational changes of cytochrome c (cyt c) at different temperatures and pH have been well examined so far by using various analytical methods. We have found that laser spray mass spectrometry enables much faster and more convenient monitoring of those changes of cyt c compared with other methods. The results correlated well with circular dichroism (CD) experiments under relatively acidic conditions, which destabilize the protein. Laser spray mass spectra of cyt c at various pH were obtained at different levels of laser power. Bimodal charge-state distributions of the protein were observed in laser spray mass spectra, indicating the two-state model of structural change; the lower charges correspond to the folded state, the higher charges to the unfolded state. Based on this result, the presumed denaturation curve of the protein was plotted as a function of laser power, and laser power by which 50% of the protein was assumed to be denatured, E50%, as obtained at each pH. We also examined the melting temperatures, Tm, of cyt c at various values of pH by using CD spectroscopy. The correlation coefficient between E50% and Tm for cyt c was 0.999, demonstrating an excellent correlation. Furthermore, laser spray analysis of ubiquitin, which is found to be more thermally stable than cyt c, gave a higher E50% than cyt c. These results indicate that laser spray mass spectrometry can be an extremely convenient method for probing thermal stabilities and dynamic conformational changes of proteins with subtle structural differences caused by slight changes in pH.     

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification. Here, we have investigated the electron capture dissociation (ECD) and collision-induced dissociation (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains.     

基体辅助激光解吸质谱法测定蛋白质分子量

本文叙述用自行研制成功的激光微探针飞行时间质谱仪及采用基体辅助激光解吸的新方法, 对溶菌酶、细胞色素C、肌红蛋白、胰蛋白酶、蛋白酶、白蛋白等多种蛋白质的分子量进行测定, 并对蛋白质混合物进行分析, 得一以满意的结果。此方法测定蛋白质分子量具有速度快(十分钟一个样品), 准确度高(±1%-0.1%), 灵敏度高(10^-^1^2～10^-^1^5mol)等优点, 是传统生物方法难以比拟的。     

Identification of peroxisomal membrane proteins of Saccharomyces cerevisiae by mass spectrometry

The identification of peroxisomal membrane proteins is very important to understand the import mechanisms of substrates and proteins into these organelles and the pathogenesis of human peroxisomal disorders like the Zellweger Syndrom. Peroxisomal membrane proteins were identified after separation by gel electrophoresis, tryptic digestion and mass spectrometric analysis. Using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), it was possible to identify 45 proteins of isolated yeast peroxisomal membranes.     

Analysis of food proteins and peptides by chromatography and mass spectrometry

The research topics and the analytical strategies dealing with food proteins and peptides are summarized. Methods for the separation and purification of macromolecules of food concern by both high-performance liquid chromatography (HPLC) on conventional packings and perfusion HPLC are examined. Special attention is paid to novel methodologies such those based on multi-dimensional systems that comprise liquid-phase based protein separation, protein digestion and mass spectrometry (MS) analysis of food peptide and proteins. Recent applications of chromatography and MS-based techniques for the analysis of proteins and peptides in food are discussed.     

Probing high order structure of proteins by fast-atom bombardment mass spectrometry

During the past decade, numerous investigations have demonstrated that the rate at which amide hydrogens located at peptide linkages undergo isotopic exchange is a sensitive probe of the high order structure and dynamics of proteins. The present investigation demonstrates that microbore high-performance liquid chromatography (HPLC) continuous-flow fast-atom bombardment mass spectrometry (FABMS) can be used to accurately quantify deuterium located at peptide linkages in short segments of large proteins. This result is important because it demonstrates the feasibility of using mass spectrometry as a tool for studying the high order structure and dynamics of large proteins. Following a period of deuterium exchange-in, a protein was placed into slow-exchange conditions and fragmented into peptides with pepsin. The digest was analyzed by continuous-flow HPLC FABMS to determine the molecular weights of the peptides, from which the number of deuterons located at the peptide linkages could be deduced. The HPLC step was used both to fractionate the peptides according to their hydrophobicities and to remove through back-exchange all deuterium except that located at peptide amide linkages. This approach has been applied to α-crystallin, a lens protein composed of two gene products with monomer molecular weights of 20 kDa and an aggregate molecular weight approaching 1000 kDa. Results from this study show that some of the peptide amide hydrogens in αA-crystallin exchange very rapidly (k > 10 h^?1) while others exchange very slowly (k < 10^?3 h^?1). The ability not only to detect that a conformational change has occurred, but also to identify the specific regions within the protein where the change occurred, was demonstrated by measuring changes in the exchange rates within these regions as the deuterium exchange-in temperature was increased from 10 to 80 ° C.     

Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.