Laser Desorption/ionization Mass Spectrometry Analysis Research Articles

Distinct Photochemistry of Odd-Carbon PAHs from the Even-Carbon Ones During the Photoaging and Analysis of Soot.

Polycyclic aromatic hydrocarbons (PAHs) are the primary organic carbons in soot. In addition to PAHs with even carbon numbers (PAHeven), substantial odd-carbon PAHs (PAHodd) have been widely observed in soot and ambient particles. Analyzing and understanding the photoaging of these compounds are essential for assessing their environmental effects. Here, using laser desorption ionization mass spectrometry (LDI-MS), we reveal the substantially different photoreactivity of PAHodd from PAHeven in the aging process and their MS detection through their distinct behaviors in the presence and absence of elemental carbon (EC) in soot. During direct photooxidation of organic carbon (OC) alone, the PAHeven are oxidized more rapidly than the PAHodd. However, the degradation of PAHodd becomes preponderant over PAHeven in the presence of EC during photoaging of the whole soot. All of these observations are proposed to originate from the more rapid hydrogen abstraction reaction from PAHodd in the EC-photosensitized reaction, owing to its unique structure of a single sp3-hybridized carbon site. Our findings reveal the photoreactivity and reaction mechanism of PAHodd for the first time, providing a comprehensive understanding of the oxidation of PAHs at a molecular level during soot aging and highlight the enhanced effect of EC on PAHodd ionization in LDI-MS analysis.

Sputter-Coated Metal Screening for Small Molecule Analysis and High-Spatial Resolution Imaging in Laser Desorption Ionization Mass Spectrometry.

Nanoparticles are efficient matrices in laser desorption/ionization (LDI) mass spectrometry (MS), especially for the profiling or imaging of small molecules. Recently, solvent-free physical vapor desorption (PVD), or sputter coating, was adopted as a homogenous method to rapidly apply metal nanoparticles (NPs) in situ to samples prior to LDI MS or MS imaging analysis. However, there has been no systematic study comparing different metal targets for the analysis of a variety of small molecule metabolites. Here, we present a screening and optimization of various sputter-coated metals, including Ag, Au, Cu, Pt, Ni, and Ti, for LDI analysis of small molecules in both positive and negative ion modes. Optimized sputter coating is then applied to high-spatial resolution LDI mass spectrometry imaging (MSI) of maize root and seed cross-sections. Noble metals, Ag, Au, and Pt, are found to be much more efficient than transition metals and organic matrices for most small metabolites. Sputter-coated metals are efficient for neutral lipids, such as triacylglycerols and diacylglycerols, but are very inefficient for most phospholipids. Graphical Abstract ᅟ.

Multivalent Aptamer/Gold Nanoparticle–Modified Graphene Oxide for Mass Spectrometry–Based Tumor Tissue Imaging

The protein mucin1 (MUC1) is an attractive target for cancer biomarkers because it is overexpressed in most adenocarcinomas. In this study, we exploited a MUC1-binding aptamer (AptMUC1) as a targeting agent for nanoparticle-based imaging systems coupled with laser desorption/ionization mass spectrometry (LDI-MS). We found that AptMUC1-conjugated gold nanoparticles immobilized, through hydrophobic and π–π interactions, on graphene oxide (AptMUC1–Au NPs/GO) bound effectively to MUC1 units on tumor cell membranes. The ultrahigh density and high flexibility of AptMUC1 on the GO surface enhanced the platform’s cooperative and multivalent binding affinity for MUC1 on cell membranes. After we had labeled MUC1-overexpressing MCF-7 cells (human breast adenocarcinoma cell line) with AptMUC1–Au NPs/GO, we used LDI-MS to monitor Au cluster ions ([Aun]+; n = 1–3), resulting in the detection of as few as 100 MCF-7 cells. We also employed this AptMUC1–Au NPs/GO–LDI-MS system to analyze four different MUC1 expression cell lines. In addition, the AptMUC1–Au NPs/GO platform could be used further as a labeling agent for tumor tissue imaging when coupled with LDI-MS. Thus, Apt–Au NPs/GO can function as a highly amplified signal transducer through the formation of large Au clusters ions during LDI-MS analysis.

High sensitive matrix-free mass spectrometry analysis of peptides using silicon nanowires-based digital microfluidic device

We present for the first time an electrowetting on dielectric (EWOD) microfluidic system coupled to a surface-assisted laser desorption-ionization (SALDI) silicon nanowire-based interface for mass spectrometry (MS) analysis of small biomolecules. Here, the transfer of analytes has been achieved on specific locations on the SALDI interface followed by their subsequent mass spectrometry analysis without the use of an organic matrix. To achieve this purpose, a device comprising a digital microfluidic system and a patterned superhydrophobic/superhydrophilic silicon nanowire interface was developed. The digital microfluidic system serves for the displacement of the droplets containing analytes, via an electrowetting actuation, inside the superhydrophilic patterns. The nanostructured silicon interface acts as an inorganic target for matrix-free laser desorption-ionization mass spectrometry analysis of the dried analytes. The proposed device can be easily used to realize several basic operations of a Lab-on-Chip such as analyte displacement and rinsing prior to MS analysis. We have demonstrated that the analysis of low molecular weight compounds (700 m/z) can be achieved with a very high sensitivity (down to 10 fmol μL(-1)).

The Opportunistic Pathogen Toxoplasma gondii Deploys a Diverse Legion of Invasion and Survival Proteins

Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered, and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins by using two complementary methodologies, two-dimensional electrophoresis/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by two-dimensional electrophoresis revealed approximately 100 spots, most of which were successfully identified by protein microsequencing or matrix-assisted laser desorption ionization-mass spectrometry analysis. Many proteins were present in multiple species suggesting they are subjected to substantial post-translational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products, including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein, and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. These findings provided a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection.

Expression and Characterization of a Murine Enzyme Able to Cleave β-Carotene: THE FORMATION OF RETINOIDS

Because animals are not able to synthesize retinoids de novo, ultimately they must derive them from dietary provitamin A carotenoids through a process known as carotene cleavage. The enzyme responsible for catalyzing carotene cleavage (beta-carotene 15,15'-dioxygenase) has been characterized primarily in rat intestinal scrapings. Using a recently reported cDNA sequence for a carotene cleavage enzyme from Drosophila, we identified a cDNA encoding a mouse homolog of this enzyme. When the cDNA was expressed in either Escherichia coli or Chinese hamster ovary cells, expression conferred upon bacterial and Chinese hamster ovary cell homogenates the ability to cleave beta-carotene to retinal. Several lines of evidence obtained upon kinetic analyses of the recombinant enzyme suggested that carotene cleavage enzyme interacts with other proteins present within cell or tissue homogenates. This was confirmed by pull-down experiments upon incubation of recombinant enzyme with tissue 12,000 x g supernatants. Matrix-assisted laser desorption ionization-mass spectrometry analysis of pulled-down proteins indicates that an atypical testis-specific isoform of lactate dehydrogenase associates with recombinant carotene cleavage enzyme. mRNA transcripts for the carotene cleavage enzyme were detected by reverse transcription-polymerase chain reaction in mouse testes, liver, kidney, and intestine. In situ hybridization studies demonstrated that carotene cleavage enzyme is expressed prominently in maternal tissue surrounding the embryo but not in embryonic tissues at 7.5 and 8.5 days postcoitus. This work offers new insights for understanding the biochemistry of carotene cleavage to retinoids.

Mechanistic Studies of 9-Ethynylphenanthrene-Inactivated Cytochrome P450 2B1

The mechanism of inactivation of the major phenobarbital-inducible cytochrome P450 of rat liver, P450 2B1, by 9-ethynylphenanthrene (9EPh) has been investigated. Matrix-assisted laser desorption ionization–mass spectrometry analysis of the cyanogen bromide-generated peptides from 9EPh-inactivated P450 2B1 confirmed the addition of a phenanthrylacetyl group to the peptide corresponding to residues 290 to 314. When this peptide was further digested with pepsin, the site of attachment could be assigned to one of the amino acids in the peptide Phe297 to Leu307 [Roberts, E. S., Hopkins, N. E., Zaluzec, E. J., Gage, D. A., Alworth, W. L., and Hollenberg, P. F. (1995)Arch. Biochem. Biophys.323, 000–000]. The inactivation by 9EPh resulted in a 90–95% loss in the NADPH-supported deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC), but had no effect on the iodosobenzene- or cumene hydroperoxide-supported metabolism of EFC. The loss of NADPH-supported activity was not affected by the addition of cytochrome b5or the presence of excess levels of reductase. The magnitude of the Type 1 spectral change upon the addition of benzphetamine was decreased with the 9EPh-modified protein. There was no decrease in the ability of modified 2B1 to form the steady-state level of the CO-reduced complex either enzymatically with NADPH and reductase or chemically with sodium dithionite, but the rate of reduction by reductase under anaerobic conditions was 57% that of native protein in the absence of substrate and 35% that of native protein in the presence of substrate. The 9EPh-modified 2B1 had an overall slower rate of NADPH oxidation, H2O2formation, and formaldehyde formation during metabolism of benzphetamine compared to native 2B1. The ratio of H2O2to HCHO was 1.0:1.0 for the native and 1.6:1.0 for the modified protein. The ability of the modified protein to form the steady-state level of the oxygen–iron complex in the presence of cyclohexane was decreased. These results are consistent with the idea that the covalent modification of one of the residues in the peptide Phe297 to Leu307 by the phenanthrylacetyl group impairs the reduction of P450 2B1 by reductase and also causes the uncoupling of NADPH utilization and oxygen consumption from product formation.