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Binding of a trinuclear platinum drug and nucleic acid conformation consequences on it are discussed. BBR3464 is the first in a new class of anticancer drugs to make it to phase I clinical trials. Its binding to different nucleic acid conformations suggests the possibility that adducts of single-strand DNA and RNA may have a role in different antitumor actions of the novel drug vs those of cisplatin.

The periodontal disease pathogen Porphyromonas gingivalis forms black-pigmented colonies on blood agar. Pigmentation is thought to be caused by the accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocyte hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases of P. gingivalis bind and degrade erythrocytes. It was observed that mutations eliminating activity of the Lys-X-specific cysteine protease, Kgp, is caused by the loss of black pigmentation of P. gingivalis W83.

A segment of the 16S/23S rDNA interspace region of a Bacillus cereus strain examined through polymerase chain reaction (PCR) amplification created several products. Upon examination of the PCR products using the electrospray ionization-Fourier transform ion cyclotron resonance technique, it was observed that a T and an A has disappeared from the coding and noncoding strand respectively. In addition, the existence of unreported sequence variations between rRNA operons in the organism is observed.

Two types of nanoscale metallocyclic molecular squares are formed via spontaneous self-assembly of angular corner units with diverse bimetallic linear linkers. The linkers, in turn, were derived via dative bonding and coordination motif. The first kind uses alkynylpyridine-containing angular building blocks as shape defining and -directing units. The second type uses bis(heteroaryliodonium) salts as corner units. Mass spectral data establish the tetrameric nature and exact structure of the molecules. Their sizes were found to fall within the range of true nanoscale supramolecular species.

Mass spectrometry has progressed a lot and had great impact in biomedicine and biochemistry. Theory, principles, instrumentation and some applications of Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be useful knowledge for teachers of chemistry. Reflectron time-of-flight (TOF) mass analyzers are useful since TOF is the most common mass analyzer for ions that come from MALDI.

A study was conducted on the preparation, via self-assembly, of a set of novel supramolecular species that combine the classical covalent crown ether and calixarene macrocycles with the coordinated molecular squares. The techniques used were multinuclear NMR with elemental analyses and ESI-FTICR mass spectrometry. The findings indicate the tetrameric nature of these complexes.

A generally applicable algorithm has been developed to allow base composition of polymerase chain reaction (PCR) products to be determined from mass spectrometrically measured molecular weights and the complementary nature of DNA. Mass measurements of arbitrary precision for single-stranded DNA species are compatible with an increasingly large number of possible base compositions as molecular weight increases. For example, the number of base compositions that are consistent with a molecular weight of 35 000 is [approximately]6000, based on a mass measurement precision of 0.01%. However, given the low misincorporation rate of standard DNA polymerases, mass measurement of both of the complementary single strands produced in the PCR reduces the number of possibilities to less than 100 at 0.01% mass precision, and base composition is uniquely defined at 0.001% mass precision. Taking into account the low misincorporation rate of standard DNA polymerases and the fact that the final PCR product also contains primers of known sequence (generally 15-20-mer in size, which flank the targeted region), this reduces the number of possible base combinations to only [approximately]3 at MW = 35 000. In addition, the number of base pairs (i.e., length of the DNA molecule) is uniquely defined. We show that the use of modified bases in PCR or post-PCR modification chemistry allows unique solutions for the base composition of the PCR product with only modest mass measurement precision.

A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described. PCR amplification utilized the DNA polymerase from Pyrococcus furiosus (Pfu) which, unlike Taq, does not incorporate a nontemplated terminal deoxyadenosine phosphate. An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus subtilis. PCR involves polymerization of nucleotide precursors using two oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions. Mass spectrometric analysis greatly benefits from removal of the oligonucleotide primers (15- and 17-mers in this instance) and nucleotide precursors since they adversely affect sensitivity and metal ion adduction results in an inaccurate molecular weight determination. In the presence of guanidinium hydrochloride, the PCR products bind preferentially to a silica resin, allowing removal of other components (i.e., dNTP's, primers, and salts). Further removal of metal ions was accomplished using a microdialysis device, allowing samples to be pumped through a hollow cellulose fiber with an external countercurrent flow of 2.5 mM ammonium acetate. Prior to injection into the mass spectrometer, the sample buffer was adjusted to 50 vol % acetonitrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal intensity. The molecular weights of the PCR products determined by nucleotide sequence and MS analysis were in excellent agreement, and several PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned. These assignments were possible due to the high mass precision, accuracy, and resolution FTICR inherently affords. This constitutes the first report demonstrating the ionization and detection of PCR products by mass spectrometry with mass precision and accuracy for assignment of such modifications or substitutions.

The combination of capillary electrophoresis (CE) with electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) has recently been demonstrated. When CE is combined with TOF-MS using an electrospray interface, the method provides fast, high-resolution separations with rapid mass analysis and the potential for very high sensitivity. In analyzing the data, one first reconstructs an electropherogram from the data set and then identifies peaks in the mass spectra. The mass spectra corresponding to the peaks in the electropherogram are then inspected to enable solute identification. However, background noise often prevents detection of peaks in the reconstructed electropherogram, thus interfering with the analysis. In this work we demonstrate alternative electropherogram reconstruction techniques that enhance its signal-to-noise ratio, allowing more immediate identification of the number of components in a mixture and their corresponding retention times. The techniques described here should also be adaptable for on-line analysis of CE-ESI-TOF data and the combination with other separation techniques coupled to mass spectrometry (e.g., LC/MS), as well as any multichannel detection scheme.

Analytical methods have been developed for the detection and quantification of cyclosporin A (CsA) in blood using time-of-flight secondary-ion mass spectrometry (TOF-SIMS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). Linear calibration curves were obtained for both methods in the range 25-2000 ng/mL in whole blood. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 7 and 23 ng/mL, respectively, for the TOF-SIMS method; the LOD and LOQ were 10 and 33 ng/mL, respectively, for the MALDI/TOF-MS method. Coefficients of variation ranged from 3 to 5% for the TOF-SIMS method and from 4 to 8% for the MALDI/TOF-MS method. Blood samples were also analyzed by a nonspecific method, fluorescence polarization immunoassay (FPIA), and by a specific method, high-performance liquid chromatography (HPLC). The TOF-MS results are in good agreement with the HPLC results, but not with the FPIA results. The TOF-MS methods can also provide information about CsA metabolites.

Rationale: Unsaturated fatty acids (UFAs) play vital roles in regulating cellular functions. In-depth structural characterization of UFAs such as localizing carbon-carbon double bonds is fundamentally important but poses considerable challenges in mass spectrometry (MS) given that the most widely accessible ion activation method, low-energy collision-induced dissociation (CID), primarily generates uninformative fragments (e.g., neutral loss of CO2) that are not suggestive of the double-bond positions. Methods: m-Chloroperoxybenzoic acid (mCPBA) was uniformly deposited onto the sample slides using a TM Sprayer, converting the carbon-carbon double bonds into epoxides under ambient conditions. The epoxidation product was ionized in situ by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry (IR-MALDESI-MS), and subsequently cleaved via CID, generating a diagnostic ion pair associated with the double-bond position. The reaction efficiency, sensitivity and relative quantification capability of the method were validated with five UFA standards dried on glass slides, and then this strategy was demonstrated on thin tissue sections of rat liver and human bladder. Results: The mCPBA reaction yielded conversion rates in the range of 44-60% in 10 min with high specificity and sensitivity. Further tandem mass spectrometry (MS/MS) of the mono-epoxidized products generated informative fragment ions specific to the double-bond positions, and relative quantification of positional isomers in binary mixtures was performed across a wide mole fraction from 0 to 1. An innovative spiral scan pattern was utilized during data acquisition, elucidating the major isomeric compositions of multiple UFAs from a tissue section in a single run. Conclusions: The on-tissue mCPBA epoxidation was implemented into an ambient MS imaging workflow to offer a rapid and simple way for in situ identification and relative quantification of double-bond positional isomers without the requirement for instrument modification. The method can be readily implemented on many other MS platforms to reveal the role of double-bond positional isomers in lipid biology and to discover potential biomarkers. [ABSTRACT FROM AUTHOR]

To better understand cell-to-cell heterogeneity, advanced analytical tools are in a growing demand for elucidating chemical compositions of each cell within a population. However, the progress of single-cell chemical analysis has been restrained by the limitations of small cell volumes and minute cellular concentrations. Here, we present a rapid and sensitive method for investigating the lipid profiles of isolated single cells using infrared matrix-assisted laser desorption electrospray ionization mass spectrometry (IR-MALDESI-MS). In this work, HeLa cells were dispersed onto a glass slide, and the cellular contents were ionized by IR-MALDESI and measured using a Q-Exactive HF-X mass spectrometer. Importantly, this approach does not require extraction and/or enrichment of analytes prior to MS analysis. Using this approach, 45 distinct lipid species, predominantly phospholipids, were detected and putatively annotated from the single HeLa cells. The proof-of-concept study demonstrates the feasibility and efficacy of IR-MALDESI-MS for rapid lipidomic profiling of single cells, which provides an important basis for future work on differentiation between normal and diseased cells at various developmental states, which can offer new insights into cellular metabolic pathways and pathological processes. Although not yet accomplished, we believe this approach can be readily used as an assessment tool to compare the number of identified species during source evolution and method optimization (intra-laboratory), and also disclose the complementary nature of different direct analytical approaches for the coverage of different types of endogenous analytes (inter-laboratory). Graphical abstract [ABSTRACT FROM AUTHOR]

The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods have been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, reaction condition length and tag concentration. Optimization of the modified method resulted in 20–100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared with the standard method. Furthermore, the identification of low-abundance glycans, such as (Fuc)1(Gal)2(GlcNAc)4(Man)3(NeuAc)1 and (Gal)3(GlcNAc)5(Man)3(NeuAc)3, was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC. [ABSTRACT FROM AUTHOR]

Chemical exposures can adversely impact fetal development. For many compounds, including common flame retardants, the mechanisms by which this occurs remain unclear, but emerging evidence suggests that disruption at the level of the placenta may play a role. Understanding how the placenta might be vulnerable to chemical exposures is challenging due to its complex structure. The primary objective of this study was to develop a method for detecting placental neurotransmitters and related metabolites without chemical derivatization so changes in the abundance and spatial distribution of neurotransmitters in rat placenta following chemical exposure could be determined using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging. Without chemical derivatization, 49 neurotransmitters and their related metabolites were putatively identified in untreated rat placenta sections using mass measurement accuracy and spectral accuracy. A few neurotransmitters were less abundant in placentas that were exposed to various flame retardants and were further investigated by KEGG metabolic pathway analysis. Many of these downregulated neurotransmitters shared the same enzyme responsible for metabolism, aromaticl-amino acid decarboxylase, suggesting a mechanistic role. These data constitute a new approach that could help identify novel mechanisms of toxicity in complex tissues. [ABSTRACT FROM AUTHOR]