The Influences of A Nitrogen Atom Position in Dinucleoside 2-,3-,4-Pyridylphosphonates on Fragmentation Patterns in Electrospray Ionization Multistage Tandem Mass Spectra

2-,3-,4-Pyridylphosphonates and their phosphonothioate congeners were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MSⁿ). It was found that the fragmentation pathways of these compounds were not influenced to any detectable extent by the stereochemistry at the phosphorus centers but were sensitive to the position of a nitrogen atom in the pyridine ring of these compounds. Possible mechanisms for fragmentations of the investigated compounds are discussed in detail.

     

Biological insights from hydrogen exchange mass spectrometry

Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes. This review provides a brief overview of the HXMS method and focuses on four recent reports to highlight applications that monitor structure and dynamics of proteins and complexes, track protein folding, and map the thermodynamics and kinetics of protein unfolding at equilibrium. These case studies illustrate typical data, analysis and results for each application and demonstrate a range of biological systems for which the interpretation of HXMS in terms of structure and conformational parameters provides unique insights into function. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Enhanced anti-topoisomerase II activity by mucoadhesive 4-CBS–chitosan/poly (lactic acid) nanoparticles

In this study, the biodegradable mucoadhesive 4-carboxybenzensulfonamide chitosan (4-CBS–chitosan)/poly (lactic acid) (PLA) nanoparticles were fabricated by the electrospray ionization technique for enhancing anti-topoisomerase II (Topo II) activity. The obtained (4-CBS–chitosan/PLA)-DOX nanoparticles were characterized using SEM, particle size analyzer. We emphasis on encapsulation efficiency, in vitro drug release behavior and also performed in vitro studies of Topo II inhibitory activity using gel electrophoresis. In addition, the cytotoxicity of the 4-CBS–chitosan/PLA nanoparticles using MTT assay was also studied. The mean particle size of spherical shaped (4-CBS–chitosan/PLA)-DOX is less than 300 nm. The DOX loaded 4-CBS–chitosan/PLA composite nanoparticles produced high entrapment efficiency of 85.8% and provided the prolonged release of DOX extended to 26 days and also still had strong Topo II inhibitory activity up to 77.4%. Overall, it was shown that 4-CBS–chitosan/PLA nanoparticles could be promising carriers for controlled delivery of anticancer drugs.     

Design,synthesis, and characterization of new cyclic d,l-α-alternate amino acid peptides by capillary electrophoresis coupled to electrospray ionization mass spectrometry

The self-assembly of peptide nanotubes (PNTs) depends on the structure and chemistry of cyclic peptide (CP) monomers, having an impact on their properties, making the choice of their monomers and their characterization a great challenge. We synthesized for the first time a new set of eight original CP sequences of 8, 10, and 12 d,l-α-alternate amino acids with a controlled internal diameter from 7 to 13 Å. They present various properties (e.g., diameter, global surface charge, hydrophobicity) that can open the way to new applications. Their structure and purity were determined thanks to a capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE–ESI–MS) methodology developed for the first time for this purpose. The CPs were successfully separated in a basic hydro-organic background electrolyte (BGE, pH 8.0, H₂O/EtOH 50:50, v/v) and analyzed in MS positive mode. The effect of CP structure on electrophoretic mobility was studied, and the mass spectra were deeply analyzed. This methodology allowed verifying their purity and the absence of linear peptide precursors as well as their stability when stored over several months. Therefore, we have developed a new CE–ESI–MS methodology for the structure and purity control of interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or as pseudo sieving tools for separative purposes.     

A comparative proteomic analysis of skin secretions of the tammar wallaby (Macropus eugenii) and the wombat (Vombatus ursinus)

The secretome of the pouch skin of the model marsupial the tammar wallaby, Macropus eugenii has been investigated using techniques of two-dimensional gel electrophoresis, in-gel trypsin digestion followed by nanoliquid chromatography coupled tandem mass spectrometry (LC-MS/MS). Differences in the patterns of secreted proteins were observed in the female pouch at three stages of maturity — reproductively immature; reproductively mature and active and mature, postreproductively active. Skin from the underarm area of mature females had a markedly different secreted protein profile. The greatest diversity of proteins was seen in the mature reproductive pouch and from an opportunistic sample collected from the pouch another mature female marsupial, the common wombat, Vombatus ursinus. A total of 20 proteins were confidently identified from the pouch skin secretions of the tammar wallaby and wombats, whilst 20 proteins were tentatively identified. In all skin secretomes, globins were the most abundant proteins whilst the antimicrobial, dermcidin was detected in the wombat sample. Some proteins such as keratin and actin could be sourced to sloughed and degraded skin cells. A number of proteins were present at such low concentrations that confident identification was not possible. This was compounded by the lack of a comprehensive database of marsupial proteins which constrains the reliability of automated identification protocols.     

Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry

A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1-2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.     

Comparison of folate quantification in foods by high-performance liquid chromatography-fluorescence detection to that by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry

A comparison study on folate quantitation was carried out between the recently developed stable isotope dilution assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the frequently used HPLC with fluorimetric detection (LC-FD). By applying LC-MS-MS, spinach, wheat bread, beef, and blood plasma were found to contain 159.2, 19.8, 1.2, and 5.6 microg/100 g total folates, respectively, whereas the respective quantitative data obtained by LC-FD were 95.5, 16.2, 0.7, and 6.8 microg/100 g. In all samples, LC-MS-MS revealed superior selectivity and precision and circumvented the shortcomings of conventional LC techniques, i.e., ambiguous peak assignment as well as high detection limits for 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid. The affinity chromatography columns used in this study showed excellent cleanup performance and permitted detection limits as low as 0.1, 0.5, 0.1, 0.08, and 0.1 microg/100 g for tetrahydrofolate (H(4)folate), 5-methyl-H(4)folate, 5-formyl-H(4)folate, 10-formylfolate, and pteroylglutamic acid, respectively. Thus, a 10-fold higher sensitivity compared to solid-phase anion-exchange cartridges was achieved. However, affinity chromatography columns revealed a significantly higher affinity toward the natural vitamers than to the racemic isotopomeric standards, which has to be considered when applying the latter in stable isotope dilution assays.     

Synthesis and characterisation of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins: application to the complexation of acyclovir

The synthesis of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins was achieved according to the standard protection-deprotection procedure. The formation of inclusion complexes between the amphiphilic alpha-, beta- and gamma-cyclodextrins and an antiviral molecule, acyclovir (ACV) was investigated by UV-visible spectroscopy (UV-Vis) and electrospray ionisation mass spectrometry (ESIMS). UV-Vis spectroscopy allowed determination of the stoichiometry and stability constants of complexes, whereas ESIMS, a soft ionisation technique, allowed the detection of the inclusion complexes. The results showed that the non-sulfated amphiphilic cyclodextrins exhibit a 1:2 stoichiometry with acyclovir, while sulfated amphiphilic cyclodextrins, except gamma-cyclodextrin, exhibit a 1:1 stoichiometry indicating the loss of one interaction site. Non-covalent interactions between acyclovir and non-sulfated amphiphilic cyclodextrins appear to take place both in the cavity of the cyclodextrin and inside the hydrophobic zone generated by alkanoyl chains. In contrast, in the case of sulfated amphiphilic cyclodextrins, the interactions appear to involve only the hydrophobic region of the alkanoyl chains.     

MS/MS profiling of taxoids from the needles of Taxus wallichiana

Ammonium cationisation has been used for taxoid profiling of partially purified methanolic extracts of needles of Taxus wallichiana growing in different regions of the Himalayas (Kashmir, Himachal Pradesh, UP Hills, Darjeeling, Sikkim and Arunachal Pradesh) by electrospray ionisation tandem mass spectrometry (MS/MS). The MS/MS spectra of the [M + NH4]+ or [M + H]+ ions gave structurally diagnostic fragment ions which revealed information about the taxane skeleton as well as the number and nature of the substituents. The rearranged 11(15-->1)-abeo-taxanes showed a characteristic elimination of the hydroxyisopropyl group with an acetoxy/benzoyloxy group from C-9. The identification of the taxoids was achieved by comparison of the MS/MS spectra with those of authentic taxoids or was based on biogenetic grounds. The results were corroborated by liquid chromatography-MS analysis. Out of the 50 taxoids identified, 21 belonged to the rearranged class. The presence of paclitaxel in the samples from four regions was confirmed: the study also revealed the occurrence of several basic taxoids in these samples. MS/MS profiling by electrospray ionisation was shown to be a fast and reliable technique for the analysis of taxoid samples.     

Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.