Alexey Kononikhin

Simple atmospheric hydrogen/deuterium exchange method for enumeration of labile hydrogens by electrospray ionization mass spectrometry.

A simple method for hydrogen/deuterium exchange in a standard electrospray (ESI) ionization source is presented. In this method, a D₂O droplet is placed between the ESI needle and the entrance of the mass spectrometer and thus saturation of the atmosphere with deuterated vapor in the ESI region is achieved. It was shown that full exchange of up to 23 labile acidic hydrogens with a minimal back exchange with the surrounding atmospheric water can be performed by this method.

Proteomics of exhaled breath: methodological nuances and pitfalls.

Abstract Background: The analysis of exhaled breath condensate (EBC) can be an alternative to traditional endoscopic sampling of lower respiratory tract secretions. This is a simple non-invasive method of diagnosing respiratory diseases, in particular, respiratory inflammatory processes. Methods: Samples were collected with a special device-condenser (ECoScreen, VIASYS Healthcare, Germany), then treated with trypsin according to the proteomics protocol for standard protein mixtures and analyzed by nanoflow high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Germany). Mascot software (Matrixscience) was used for screening the database NCBInr for proteins corresponding to the peptide maps that were obtained. Results: EBCs from 17 young healthy non-smoking donors were collected. Different methods for concentrating protein were compared in order to optimize EBC preparations for proteomic analysis. The procedure that was chosen allowed identification of proteins exhaled by healthy people. The major proteins in the condensates were cytoskeletal keratins. Another 12 proteins were identified in EBC from healthy non-smokers. Some keratins were found in the ambient air and may be considered exogenous components of exhaled air. Conclusions: Knowledge of the normal proteome of exhaled breath allows one to look for biomarkers of different disease states in EBC. Proteins in ambient air can be identified in the respiratory tract and should be excluded from the analysis of the proteome of EBC. The results obtained allowed us to choose the most effective procedure of sample preparation when working with samples containing very low protein concentrations. Clin Chem Lab Med 2009;47:706–12.

Conformational changes of ubiquitin during electrospray ionization as determined by in-ESI source H/D exchange combined with high- resolution MS and ECD fragmentation

In the paper, we have demonstrated the possibility of performing hydrogen/deuterium (H/D) exchange of proteins in the region of gas-phase ion formation in an electrospray ion source by saturating the electrospray ionization source with vapors of a deuterating agent (D(2)O or MeOD). In this region, charged droplets are shrinking and the protein ions transfer into the gas phase. As a model protein, we have used ubiquitin whose ion mobility spectrometry and gas-phase H/D exchange in the vacuum part of a mass spectrometer demonstrated the presence of gas-phase conformers with different cross sections and H/D exchange rates. In our experiments, we observed monomodal deuterium distributions for all solvents, charge states, desolvating capillary temperature and types of deuterating agent. Also, we found that the number of H/D exchanges increases with an increasing desolvating capillary temperature and decreasing charge state. We observed that solution composition (49 : 50 : 1 H(2)O : MeOH : formic acid or 99 : 1 H(2)O : formic acid) influences the charge-state distribution but did not change the degree of H/D exchange for the same charge state. Electron-capture dissociation fragmentation shows that higher charge states contain a segment that is protected from access by the deuterating agent.

Molecular Mapping of Sorbent Selectivities with Respect to Isolation of Arctic Dissolved Organic Matter as Measured by Fourier Transform Mass Spectrometry

The objectives of this study were to identify molecular features characteristic to arctic DOM from the Kolyma River basin and to elucidate structural imprints induced by a choice of the sorption technique. To achieve this goal, DOM was isolated from the Kolyma River basin with a use of three nonionic sorbents: Amberlite XAD-8 resin, PPL- and C18 - SPE cartridges, and one anion exchanging resin-diethylaminoethyl (DEAE) -cellulose. The structural studies were conducted with a use of electrospray ionization Fourier Transform Ion Cyclotron Resonance (ESI FT-ICR) mass spectrometry and liquid state (1)H NMR spectroscopy. The DOM isolates obtained with a use of PPL and C18 cartridges were characterized with higher content of aliphatic compounds as compared to XAD-8 and DEAE-isolates. In total, for all arctic DOM isolates we observed predominance of hydrogen saturated compounds with high H/C values of identified formulas from FT-ICR MS data. (1)H NMR spectroscopy studies have confirmed this trend and revealed high contribution of alkyl-chain protons into the spectral density of the arctic DOM reaching 43% for PPL isolates.

Enumeration of Labile Hydrogens in Natural Organic Matter by Use of Hydrogen/Deuterium Exchange Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

A method to enumerate labile hydrogens in all constituents of molecular ensemble of natural organic matter (NOM) based on our previously developed simple hydrogen/deuterium (H/D) exchange (electrospray ionization (ESI) ion source (Kostyukevich et al. Anal. Chem. 2013, 85, 5330) and ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry is presented. The method was applied for analysis of Suwannee River fulvic acid (SRFA), which is an International Humic Substances Society standard, as well as Siberian crude oil; and lignosulfonate. We found that SRFA and lignosulfonate molecules contain 2-5 labile hydrogens, and their number increases with the number of oxygens in the molecule. Also, we observed that compounds of Siberian crude oil ionizing in positive-ESI mode do not have labile hydrogens, while compounds ionizing in negative-ESI mode have one labile hydrogen that detaches during ESI ionization.

In-ESI source hydrogen/deuterium exchange of carbohydrate ions.

We present the investigation of hydrogen/deuterium (H/D) exchange of carbohydrates ions occurring in the electrospray ion source. The shape of the deuterium distribution was observed to be considerably dependent on the temperature of the ion transfer tube and the solvent used. If deuterated alcohol (EtOD or MeOD) or D2O/deuterated alcohol is used as an electrospray solvent, then for high temperatures (>350 °C), intensive back exchange is observed, resulting in ∼30% depth of the deuterium exchange. At low temperatures (<150 °C), the back exchange is weaker and the depth of the deuterium exchange is ∼70%. In the intermediate temperature region (∼250 °C), the deuterium distribution is unusually wide for methanol and bimodal for ethanol. The addition of 1% formic acid results in low (∼30%) depth of the deuterium exchange for any temperature in the operating region. The bimodal distribution for the ethanol can be possibly explained by the presence of differently folded gas-phase ions of carbohydrates.

Mass spectrometric characterization of photooxidative protein modifications in Arabidopsis thaliana thylakoid membranes

Oxidative and nitrosative stress leaves footprints in the plant chloroplast in the form of oxidatively modified proteins. Using a mass spectrometric approach, we identified 126 tyrosine and 12 tryptophan nitration sites in 164 nitrated proteolytic peptides, mainly from photosystem I (PSI), photosystem II (PSII), cytochrome b(6) /f and ATP-synthase complexes and 140 oxidation products of tyrosine, tryptophan, proline, phenylalanine and histidine residues. While a high number of nitration sites were found in proteins from four photosynthetic complexes indicating that the nitration belongs to one of the prominent posttranslational protein modifications in photosynthetic apparatus, amino acid oxidation products were determined mostly in PSII and to a lower extent in PSI. Exposure of plants to light stress resulted in an increased level of tyrosine and tryptophan nitration and tryptophan oxidation in proteins of PSII reaction center and the oxygen-evolving complex, as compared to low light conditions. In contrast, the level of nitration and oxidation of these amino acid residues strongly decreased for all light-harvesting proteins of PSII under the same conditions. Based on these data, we propose that oxidative modifications of proteins by reactive oxygen and nitrogen species might represent an important regulatory mechanism of protein turnover under light stress conditions, especially for PSII and its antenna proteins.

Simple Synthesis of Ruthenium π Complexes of Aromatic Amino Acids and Small Peptides

The interaction of [Ru(eta(6)-C(10)H(8))(Cp)](+) (Cp=C(5)H(5)) with aromatic amino acids (L-phenylalanine, L-tyrosine, L-tryptophane, D-phenylglycine, and L-threo-3-phenylserine) under visible-light irradiation gives the corresponding [Ru(eta(6)-amino acid)(Cp)](+) complexes in near-quantitative yield. The reaction proceeds in air at room temperature in water and tolerates the presence of non-aromatic amino acids (except those which are sulfur containing), monosaccharides, and nucleotides. The complex [Ru(eta(6)-C(10)H(8))(Cp)](+) was also used for selective labeling of Tyr and Phe residues of small peptides, namely, angiotensin I and II derivatives.

Phosphorylation and nitration levels of photosynthetic proteins are conversely regulated by light stress

Using a label-free mass spectrometric approach, we investigated light-induced changes in the distribution of phosphorylated and nitrated proteins within subpopulations of native photosynthetic complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to growth light (GL) and subsequently exposed to high light (HL). Eight protein phosphorylation sites were identified in photosystem II (PSII) and the phosphorylation level of seven was regulated by HL as determined based on peak areas from ion chromatograms of phosphorylated and non-phosphorylated peptides. Although the phosphorylation of PSII proteins was reported in the past, we demonstrated for the first time that two minor antenna LHCB4 isoforms are alternately phosphorylated under GL and HL conditions in PSII monomers, dimers and supercomplexes. A role of LHCB4 phosphorylation in state transition and monomerization of PSII under HL conditions is proposed. We determined changes in the nitration level of 23 tyrosine residues in five photosystem I (PSI) and nine PSII proteins and demonstrated for the majority of them a lower nitration level in PSI and PSII complexes and supercomplexes under HL conditions, as compared to GL. In contrast, the nitration level significantly increased in assembled/disassembled PSI and PSII subcomplexes under HL conditions. A possible role of nitration in (1) monomerization of LHCB1-3 trimers under HL conditions (2) binding properties of ferredoxin-NADP+ oxidoreductase to photosystem I, and (3) PSII photodamage and repair cycle, is discussed. Based on these data, we propose that the conversely regulated phosphorylation and nitration levels regulate the stability and turnover of photosynthetic complexes under HL conditions.

In ESI-source H/D exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa: the role of the temperature of the desolvating capillary on H/D exchange

Transition of proteins from the solution to the gas phase during electrospray ionization remains a challenging problem despite the large amount of attention it has received during the past few decades. One of the major questions relates to the extent to which proteins in the gas phase retain their condensed phase structures. We have used in-electrospray source hydrogen/deuterium exchange to determine the number of deuterium incorporations as a function of protein mass, charge state and temperature of the desolvating capillary where the reaction occurs. All experiments were performed on a Thermo LTQ FT Ultra equipped with a 7-T superconducting magnet. Ions were generated by an IonMax Electrospray ion source operated in the positive ESI mode. Deuterium exchange was performed by introducing a droplet of D2 O beneath the ESI capillary. We systematically investigated gas phase hydrogen/deuterium (H/D) exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa. We observed that almost all proteins demonstrate similar exchange rates for all charge states and that these rates increase exponentially with the temperature of the desolvating capillary. We did not observe any clear correlation of the number of H/D exchanges with the value of the cross section for a corresponding charge state. We have demonstrated the possibility of performing in-ESI source H/D exchange of large proteins under atmospheric pressure. The simplicity of the experimental setup makes it a useful experimental technique that can be applied for the investigation of gas phase conformations of proteins.