Hongying Zhong

Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis

The characterization of protein modifications is essential for the study of protein function using functional genomic and proteomic approaches. However, current techniques are not efficient in determining protein modifications. We report an approach for sequencing proteins and determining modifications with high speed, sensitivity and specificity. We discovered that a protein could be readily acid-hydrolyzed within 1 min by exposure to microwave irradiation to form, predominantly, two series of polypeptide ladders containing either the N- or C-terminal amino acid of the protein, respectively. Mass spectrometric analysis of the hydrolysate produced a simple mass spectrum consisting of peaks exclusively from these polypeptide ladders, allowing direct reading of amino acid sequence and modifications of the protein. As examples, we applied this technique to determine protein phosphorylation sites as well as the sequences and several previously unknown modifications of 28 small proteins isolated from Escherichia coli K12 cells. This technique can potentially be automated for large-scale protein annotation.

Typing of unknown microorganisms based on quantitative analysis of fatty acids by mass spectrometry and hierarchical clustering.

Rapid identification of unknown microorganisms of clinical and agricultural importance is not only critical for accurate diagnosis of infections but also essential for appropriate and prompt treatment. We describe here a rapid method for microorganisms typing based on quantitative analysis of fatty acids by iFAT approach (Isotope-coded Fatty Acid Transmethylation). In this work, lyophilized cell lysates were directly mixed with 0.5M NaOH solution in d3-methanol and n-hexane. After 1 min of ultrasonication, the top n-hexane layer was combined with a mixture of standard d0-methanol derived fatty acid methylesters with known concentration. Measurement of intensity ratios of d3/d0 labeled fragment ion and molecular ion pairs at the corresponding target fatty acids provides a quantitative basis for hierarchical clustering. In the resultant dendrogram, the Euclidean distance between unknown species and known species quantitatively reveals their differences or shared similarities in fatty acid related pathways. It is of particular interest to apply this method for typing fungal species because fungi has distinguished lipid biosynthetic pathways that have been targeted for lots of drugs or fungicides compared with bacteria and animals. The proposed method has no dependence on the availability of genome or proteome databases. Therefore, it is can be applicable for a broad range of unknown microorganisms or mutant species.

Chemical Imaging of Latent Fingerprints by Mass Spectrometry Based on Laser Activated Electron Tunneling

Identification of endogenous and exogenous chemicals contained in latent fingerprints is important for forensic science in order to acquire evidence of criminal identities and contacts with specific chemicals. Mass spectrometry has emerged as a powerful technique for such applications without any derivatization or fluorescent tags. Among these techniques, MALDI (Matrix Assisted Laser Desorption Ionization) provides small beam size but has interferences with MALDI matrix materials, which cause ion suppressions as well as limited spatial resolution resulting from uneven distribution of MALDI matrix crystals with different sizes. LAET (Laser Activated Electron Tunneling) described in this work offers capabilities for chemical imaging through electron-directed soft ionization. A special film of semiconductors has been designed for collection of fingerprints. Nanoparticles of bismuth cobalt zinc oxide were compressed on a conductive metal substrate (Al or Cu sticky tape) under 10 MPa pressure. Resultant uniform thin films provide tight and shining surfaces on which fingers are impressed. Irradiation of ultraviolet laser pulses (355 nm) on the thin film instantly generates photoelectrons that can be captured by adsorbed organic molecules and subsequently cause electron-directed ionization and fragmentation. Imaging of latent fingerprints is achieved by visualization of the spatial distribution of these molecular ions and structural information-rich fragment ions. Atomic electron emission together with finely tuned laser beam size improve spatial resolution. With the LAET technique, imaging analysis not only can identify physical shapes but also reveal endogenous metabolites present in females and males, detect contacts with prohibited substances, and resolve overlapped latent fingerprints.

Rapid Transmethylation and Stable Isotope Labeling for Comparative Analysis of Fatty Acids by Mass Spectrometry

Fatty acids covalently bonded with other molecules have been implicated in many important biological processes. We describe here a rapid approach termed isotope-coded fatty acid transmethylation (iFAT) that integrates extraction, transmethylation, and isotopic labeling into a single step with the aid of ultrasonic irradiation for comparative analysis of fatty acids by mass spectrometry. In this approach, samples without any prefractionation were mixed with a methanol solution of 0.5 M NaOH and an n-hexane solution. The intense wave shocks and cavitations generated by ultrasonic irradiation not only speed the alkaline-catalyzed transmethylation reaction but also facilitate the simultaneous mass transfer of fatty acid methyl esters into the top n-hexane extraction phase that was injected into a GC/MS system. By using commercially available d(3)-methanol, we were able to compare the intensity of labeled and unlabeled methyl esters and their corresponding fragment ions. The detection limit can be down to the picogram level. Major advantages of the iFAT strategy are summarized in the following: (1) Efficient heterogeneous reactions. Solid samples such as dried cell lysates or detergent-resistant fractions can be readily transformed and analyzed with the aid of ultrasound irradiation. (2) Accurate quantification of fatty acids. Evaluation of the completeness or losses of transformation reactions across lipid classes has been hampered due to a lack of suitable methods. Isotope labeling can be used as an internal standard for accurate comparison of the fatty acid composition in different cell states. (3) Reduced interferences from complex biological context. The iFAT strategy not only differentially labels fatty acids in different samples, but also volatilizes those molecules, and thus, they are isolated from the bulk background and analyzed by GC/MS. This proposed approach has been applied to quantitatively determine the fatty acid composition in plant oil and in budding yeast cell lysates and detergent-resistant fractions. It should provide a widely applicable means for quantitative comparison of the fatty acid composition in cells and tissues.

Gas chromatography–mass spectrometric analysis of bonded long chain fatty acids in a single zebrafish egg by ultrasound-assisted one-step transmethylation and extraction

Changes in the level of lipid composition in a single zebrafish egg have been involved in biological responses to chemical exposures. In this paper, an one-step transmethylation of lipids and simultaneous extraction of resultant fatty acid methyl esters followed by gas chromatography-mass spectrometric analysis was developed for the identification of bonded long chain fatty acids in a single zebrafish egg. The efficiency of transmethylation under different experimental conditions has been investigated. Dried egg homogenates were directly mixed with either 0.5 M NaOH or 1% H2SO4 or 4% HCl solution of methanol and then an n-hexane solution was added on the top. Ultrasonication of this immiscible liquid-liquid-solid system produces high velocity impacts between solid particles and liquid phases and thus promotes mass transfer among phases. It was demonstrated that ultrasound irradiation has strong effect on the alkaline-catalyzed transmethylation of lipids but cannot significantly change the acid-catalyzed transmethylation of lipids. With the aid of ultrasonication, transmethylation can be combined with simultaneous extraction of the resultant fatty acid methyl esters into n-hexane phase. This approach simplifies the sample preparation procedure, shortens the reaction time but improves the efficiency of the transmethylation of lipids and reduces sample losses, especially for small size samples. It has been applied to determine bonded fatty acids in a single zebrafish egg. In total, 28 fatty acids from a single zebrafish egg have been identified reproducibly.

Measurement of Laser Activated Electron Tunneling from Semiconductor Zinc Oxide to Adsorbed Organic Molecules by a Matrix Assisted Laser Desorption Ionization Mass Spectrometer

Measurement of light induced heterogeneous electron transfer is important for understanding of fundamental processes involved in chemistry, physics and biology, which is still challenging by current techniques. Laser activated electron tunneling (LAET) from semiconductor metal oxides was observed and characterized by a MALDI (matrix assisted laser desorption ionization) mass spectrometer in this work. Nanoparticles of ZnO were placed on a MALDI sample plate. Free fatty acids and derivatives were used as models of organic compounds and directly deposited on the surface of ZnO nanoparticles. Irradiation of UV laser (λ=355 nm) with energy more than the band gap of ZnO produces ions that can be detected in negative mode. When TiO(2) nanoparticles with similar band gap but much lower electron mobility were used, these ions were not observed unless the voltage on the sample plate was increased. The experimental results indicate that laser induced electron tunneling is dependent on the electron mobility and the strength of the electric field. Capture of low energy electrons by charge-deficient atoms of adsorbed organic molecules causes unpaired electron-directed cleavages of chemical bonds in a nonergodic pathway. In positive detection mode, electron tunneling cannot be observed due to the reverse moving direction of electrons. It should be able to expect that laser desorption ionization mass spectrometry is a new technique capable of probing the dynamics of electron tunneling. LAET offers advantages as a new ionization dissociation method for mass spectrometry.

Magnetic nanoparticles of nitrogen enriched carbon (mnNEC) for analysis of pesticides and metabolites in zebrafish by gas chromatography-mass spectrometry.

Nanosized carbon based sorbents have been widely used for separation, enrichment and desalting of biological samples because of their distinguished characteristics. In this work, magnetic nanoparticles of nitrogen enriched carbon (mnNEC) have been developed for enrichment of organochlorine pesticide DDT and metabolite DDE that have been accumulated in zebrafish during the course of environmental exposure. Polymerization of pyrrole was performed in the aqueous suspension of Fe(3)O(4) nanoparticles. Resultant core-shell nanoparticules coated with polypyrrole were then subjected to a process of carbonization under high temperature and nitrogen atmospheric condition. The presence of nitrogen atoms in carbon nanoparticles increases the hydrophilicity and dispersability in aqueous samples. It has been experimentally demonstrated that mnNEC can be effectively dispersed in aqueous samples and rapidly isolated by the application of an external magnetic field. Recoveries of DDT and DDE from water range from 90% to 102% and 85-97% respectively. In combination with Selected Ion Monitoring (SIM) experiments of gas chromatography-mass spectrometry, the detection limit can be down to low ng/mL level. By using mnNEC approach, two interesting results have been found for zebrafish with 60 days exposure to DDT (1 μg/l). (1) There is higher concentration of DDT (37-143 ng/g) and DDE (173-1108 ng/g) in male zebrafish body tissues than that of female (7-52 ng/g and 146-362 ng/g for DDT and DDE respectively). (2) There is high ratio of DDE/DDT for both female and male zebrafish, implying high environmental persistence and ongoing bioaccumulation.

Comparative analysis of S-fatty acylation of gel-separated proteins by stable isotope-coded fatty acid transmethylation and mass spectrometry

Covalent attachment of palmitic acid or other fatty acids to the thiol groups of cysteine residues of proteins through reversible thioester bonds has an important role in the regulation of diverse biological processes. We describe here the development of a mass spectrometry protocol based on stable isotope–coded fatty acid transmethylation (iFAT) for qualitative and comparative analysis of protein S-fatty acylation under different experimental conditions. In this approach, cellular proteins extracted from different cell states are separated by SDS-PAGE and then the gel is stained with either Coomassie blue or Nile red for improved sensitivity. Protein bands are excised and then an in-gel stable iFAT procedure is performed. The fatty acid methyl esters resulting from derivatization with d0- and d3-methanol are identified by mass spectrometry. By measuring the intensities of labeled and unlabeled fragment ion pairs of fatty acid methyl esters, the levels of S-fatty acylation in different cells or tissues can be compared. This approach has been applied to monitor the changes of S-fatty acylation of zebrafish liver proteome in response to environmental dichlorodiphenyltrichloroethane exposure. Compared with the approach using metabolic incorporation of radioactive fatty acid analogs, it is not only simple and effective but also eliminates the hazards of handling radioactive isotopes.

Mass spectrometric monitoring of interfacial photoelectron transfer and imaging of active crystalline facets of semiconductors

Monitoring of interfacial electron transfer (ET) in situ is important to understand the ET mechanism and designing efficient photocatalysts. We describe herein a mass spectrometric approach to investigate the ultrafast transfer of photoelectrons that are generated by ultraviolet irradiation on surfaces of semiconductor nanoparticles or crystalline facets. The mass spectrometric approach can not only untargetedly detect various intermediates but also monitor their reactivity through associative or dissociative photoelectron capture dissociation, as well as electron detachment dissociation of adsorbed molecules. Proton-coupled electron transfer and proton-uncoupled electron transfer with radical initiated polymerization or hydroxyl radical abstraction have been unambiguously demonstrated with the mass spectrometric approach. Active crystalline facets of titanium dioxide for photocatalytic degradation of juglone and organochlorine dichlorodiphenyltrichloroethane are visualized with mass spectrometry imaging based on ion scanning and spectral reconstruction. This work provides a new technique for studying photo-electric properties of various materials.

Imaging of Endogenous Metabolites of Plant Leaves by Mass Spectrometry Based on Laser Activated Electron Tunneling

A new mass spectrometric imaging approach based on laser activated electron tunneling (LAET) was described and applied to analysis of endogenous metabolites of plant leaves. LAET is an electron-directed soft ionization technique. Compressed thin films of semiconductor nanoparticles of bismuth cobalt zinc oxide were placed on the sample plate for proof-of-principle demonstration because they can not only absorb ultraviolet laser but also have high electron mobility. Upon laser irradiation, electrons are excited from valence bands to conduction bands. With appropriate kinetic energies, photoexcited electrons can tunnel away from the barrier and eventually be captured by charge deficient atoms present in neutral molecules. Resultant unpaired electron subsequently initiates specific chemical bond cleavage and generates ions that can be detected in negative ion mode of the mass spectrometer. LAET avoids the co-crystallization process of routinely used organic matrix materials with analyzes in MALDI (matrix assisted-laser desorption ionization) analysis. Thus uneven distribution of crystals with different sizes and shapes as well as background peaks in the low mass range resulting from matrix molecules is eliminated. Advantages of LAET imaging technique include not only improved spatial resolution but also photoelectron capture dissociation which produces predictable fragment ions.