Current Analytical Chemistry - Volume 9, Issue 2, 2013

Amyloid structures accumulate and propagate through self-assembly of partially folded proteins and peptides, resulting in a range of disease states. Key to understanding amyloid disease is the characterisation of the often toxic oligomeric species formed during the early stages of fibril assembly. Electrospray ionisation- ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) has emerged as a powerful tool to investigate amyloid oligomer assembly and protein conformation change. In this review we focus on the role of ESI-IMS-MS in understanding and probing conformational changes and the early stages of protein aggregation.

Intrinsically disordered proteins do not adopt well-defined native structures and therefore present an intriguing challenge in terms of structural elucidation as they are relatively inaccessible to traditional approaches such as NMR and X-ray crystallography. Many members of this important group of proteins have a distinct biological function and frequently undergo a conformational change on binding to their physiological targets which can in turn modulate their function. Furthermore, many intrinsically unstructured proteins are associated with a wide range of major diseases including cancer and amyloid-related disorders. Here, electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESIIMS- MS) has been used to probe the conformational characteristics of two intrinsically disordered proteins: apocytochrome c and apo-osteocalcin. Both proteins are structured in their holo-states when bound to their respective substrates, but disordered in their apo-states. Here, the conformational properties of the holo- and the apo-protein forms for both species have been analysed and their mass spectral data and ion mobility spectrometry-derived collision crosssectional areas, indicative of their physical size, compared to study the relationship between substrate binding and tertiary structure. In both cases, the intrinsically unstructured apo-states populated multiple conformations with larger crosssectional areas than their holo-analogues, suggesting that intrinsic disorder in proteins does not preclude the formation of preferred conformations. Additionally, analysis of truncated analogues of osteocalcin has located the region of the protein responsible for the conformational changes detected upon metal cation binding. Together, the data illustrate the scope and utility of ESI-IMS-MS for studying the characteristics and properties of intrinsically disordered proteins whose analysis by other techniques is limited.

Field asymmetric waveform ion mobility spectrometry (FAIMS) is a gas-phase atmospheric pressure separation technique that exploits the difference in the mobility of an ion in alternating low and high electric fields applied between two closely spaced electrodes. FAIMS has the potential to simplify mass spectra by separating complex mixtures of ions, including isobaric ions, prior to mass spectral detection. It is a technique that is distinct from both chromatographic and mass-to-charge (m/z) separations. This mini-review discusses the principles of FAIMS separations and instrumentation and the application of FAIMS combined with mass spectrometry for the analysis of peptides and proteins.

The use of gas-phase ion mobility separation in LC-MS based bottom-up proteomic experiments is described. A data-independent acquisition approach was employed in combination with one and two dimensional reversed phase liquid chromatography separations. The ion mobility component in the acquisition schema affords additional acquisition specificity and improved peptide resolution in terms of system peak capacity. The additional acquisition specificity was mainly achieved by means of the combined retention and drift time association of product to precursor ions and chemical noise reduction. Experimental data from replicate experiments were utilized to determine the rate of increase for the number of detected ions and deconvoluted accurate mass – retention time pairs and protein and peptide identifications as a measure of peak capacity by varying mass spectral resolution, consideration of multidimensional separations and the incorporation of ion mobility into the analytical schema. The presented data reveals a high degree of reproducibility at the ion detection, peptide and protein level, when analyzed under identical conditions, with a concurrent average increase in the number of identified peptides and proteins of 160% in the instance of mobility assisted acquisitions. Two-dimensional separations independently improved the peptide and protein identification rates by another 260% and 320%, respectively. Collectively, both separation techniques, reversed phase chromatography and ion mobility, increased the peak capacity and number of peptides and proteins significantly. The applicability of the technique and instrument platform for qualitative and quantitative proteomics studies will be demonstrated.

Hypoxia is a common feature observed in solid tumours. It is a target of interest in oncology as it has been found to be closely associated with tumour progression, metastasis and aggressiveness and confers resistance to a variety of chemotherapeutic agents as well as radiotherapy. AQ4N, also known as banoxatrone or 1,4-bis-[2-(dimethylamino-Noxide) ethyl]amino-5,8-dihydroxyanthracene-9,10-dione is a very promising bioreductive prodrug. This paper, describes an application of MALDI-MSI combined with ion mobility separation and an "on-tissue" bottom up proteomic strategy to obtain proteomic data from AQ4N dosed tumour xenograft tissue sections. These data are then correlated with the drug distribution determined also using MALDI-ion mobility separation-mass spectrometry imaging (MALDI-IMS-MSI). PCA-DA and OPLS-DA have been used to compare treated and untreated xenografts and of note is the marked increase in expression of Histone H3.

A metallomic analytical approach based on the use of size exclusion chromatography (Superdex-75 and Superdex TM-Peptide) coupled to ICP-MS has been used to obtain metal profiles related to metallobiomolecules overexpression or inhibition in mice caused by contamination. The method is complemented with a second chromatographic dimension (anion and cation exchange chromatography) for species purification and isolation, and in some cases metal-species identification by mass spectrometry. The biological response of free-living mice Mus spretus (liver, kidney and brain) from contaminated and non-contaminated areas of areas of Doñana National Park (southwest Spain) and the surroundings were found to be affected by differential contamination. The results reveal the presence of high concentration of Fe, Cu and Zn in liver and As and Cd in kidney in contaminated areas. Upregulation of peaks traced by Cu, Zn and Cd at 7 kDa (matching with metallothionein I standard) in liver cytosolic extract is triggered by the presence of contaminants. Other Cu and Zn-peaks located at 32 kDa are also overexpressed by pollution and can be related to upregulation of superoxide dismutase. In kidney the presence of Cu, Zn and Cd-binding peaks located at 7 kDa from sites affected by contamination can also be observed. Finally, the abundance of low molecular mass As species in kidney due to the excretion of this element as methyl and dimethylarsenic is remarkable. In addition, the laboratory mice Mus musculus was used as control due to already sequenced and genetically homologous to Mus spretus. For this reason, biological response of Mus musculus to metal contamination was checked by exposure experiments to Cd and As and results were compared with those obtained from Mus spretus from contaminated areas.

Organic germanium compounds, such as bis-beta-carboxyethylgermanium sesquioxide (“Ge-132”), have attained widespread distribution – particularly in self-medication – and are applied as tonics due to their presumed anticancer activities. In general, the toxicity of this particular organo-germanium compound is low. In contrast to this, acute and chronic toxic effects of inorganic germanium dioxide (GeO2) have been demonstrated. In this paper, chloride-generation headspace solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is proposed as a technique for the determination of trace amounts of GeO2 in Ge-132. This method takes advantage of the fact that Ge-132 is not able to generate volatile species upon reaction with conc. HCl. The conditions for the generation of germanium chloride (GeCl4), such as SPME fibre selection, extraction time and HCl concentration were investigated. The detection limit was established at 0.28 μg/ml (as Ge). Thus, this approach provides a novel, simple and fast platform for the determination of inorganic Ge in organogermanium formulations.

Recently, the scientific interest for greener analytical methods, has led to the development of novel miniaturized liquid phase microextraction (LPME) methodologies resulting in a minimum amount of organic solvent used, along with fast and economical procedures. Sample preparation is the most time-consuming and polluting step in any analytical method. This review aims to provide an overview of the advances in all these unconventional liquid phase microextraction methodologies, such as single-drop microextraction (SDME), hollow-fiber liquid phase microextraction (HF-LPME), dispersive liquid-liquid microextraction (DLLME) and solidified floating organic drop (SFO) microextraction and their potential use for metal, metalloid and organometallic compounds determination as well as for metal speciation. The new trends in the field of miniaturization and automation are also presented.

In this work, the capability of a hybrid tandem technique of LCMS-IT-TOF was examined for speciation of tripropyltin (TPrT), tributyltin (TBT), tripentyltin (TPeT) and triphenyltin (TPhT) and a relatively fast and accurate method was developed for their determination in aqueous solutions. In this context, several chromatographic parameters were studied such as chromatographic column, mobile phase, flow rate and gradient program in order to optimise the separation of the above species. Optimum separation was achieved using reverse phase C18 bonded-silica column. The mobile phase consisted of water and methanol and acetic acid at a total flow rate of 0.4 mL min–1 and the separation was completed in less than 8 min. Determination of the analytes was performed based on the signal of characteristic ions for each particular triorgano-tin compound. The detection limits of the set of the above four triorgano-tin species were ranged between 13 – 45 pg as absolute mass of Sn injected into the column and the relative standard deviation was ranged between 4.3 – 9.9 %. Finally, the performance characteristics of the method were compared to those reported in literature using similar tandem LC-MS techniques.

The aim of this study was to compare two different widely-used methods for the determination of hexavalent chromium (Cr(VI)) in water samples by Electrothermal Atomic Absorption Spectrometry (ETAAS). Both methods are based on the complexation - reaction of Cr(VI) with an organic complexation reagent, which is then extracted and preconcentrated in organic solvent. In the first method, ammonium pyrrolidine dithiocarbamate (APDC) is used as complexation reagent, whereas 1,5-diphenylcarbazide (DPC) is used in the second method. The speciation methods were optimized and validated. Both methods were applied for the determination of Cr(VI) in the same multi-level groundwater samples (0.060 – 42 μg/L, n=13) and the results were compared statistically. Beside the comparison of the two extraction methods (APDC, DPC), the samples were also analyzed by Reagent Free Ion Chromatography (RFIC) with conductivity detector and statistical comparison was also performed. Paired t-test was applied and the results indicated that there was no statistically significant difference between the three methods. Useful conclusions about the analytical performance of these widely-used-in-routine-labs methods were drawn. The selectivity of Cr(VI) determination was significant for both methods. The DPC method had lower limit of detection than APDC, however the APDC method was more robust than the DPC method. Both methods are appropriate for the determination of Cr(VI) in different ground water samples at sub-μg/L levels.

A sequential-injection hydride-generation atomic absorption spectrometry (SIA-HG-AAS) system for the determination of trace levels of selenium was designed and characterised. The system included on-line pre-reduction from Se(VI) to Se(IV) and used NaBH4 as reagent for the generation of the hydride. The operation of the system was optimised regarding the main operating variables by means of appropriate experimental designs. The analytical response was linear in the range 2.0 - 20 μg L-1 (Apeak = 0.0048 C, with C = concentration in μg L-1, r2 = 0.9973). Detection (3 s) and quantification (10 s) limits were 0.6 and 2.0 μg L-1 respectively with precision (repeatability, sr(%), n = 10) from 2.2% to 3.6% for concentrations between 5 and 20 μg L-1. Sampling frequency was 60 hour-1. An interference study was carried out comparing the performance of both the proposed system and an HG-AAS reference system involving batch generation of the hydride. As, Co, Cu, Fe(II), Fe(III), Hg, Ni, nitrate and Zn were chosen as potential interferents. Interference from As and nitrate was shown to be more severe in the proposed system than in the batch one. Co, Hg and Zn did not present any significant interference in either system. The presence of Fe(III) at the concentrations tried showed the same behaviour in the response for both systems. The range of concentrations of Cu, Fe(II) and Ni allowing an interference-free selenium determination was wider in the SIA-HG-AAS system than in the reference HG-AAS system.

We demonstrate the electrochemical pretreatment of carbon fiber microelectrodes (CFEs) based on the sinusoidal- wave potential cycling (50 Hz) of CFEs prior to amperometric analysis. The surface modification and morphology of CFEs after pretreatment was studied using Raman spectroscopy and scanning electron microscopy. The pretreated CFEs were extensively tested for sensing of bioactive compounds (dopamine, nitric oxide, ascorbate and nitrite) by constant current amperometry at 830 mV vs. Ag/AgCl.

Hydrogen sulphide (H2S) was traditionally recognized as a pungent toxic gas, but this view has now been revised to include its role as an endogenously produced signaling molecule in mammalian cells, affecting various physiological and pathophysiological processes. Endogenously produced H2S in tissues and blood is reported to be lower than 15 nmol/L [1], which requires sample pre-concentration step and sensitive detection technologies. Gas chromatography (GC) equipped with a variety of detectors is regarded of the choice for quantitative detection of H2S. However, the high background moisture in mammalian breath greatly challenges the selectivity and sensitivity of this technique and dictates the need for interfacing moisture trapping techniques prior to preconcentration and detection steps. In preparation for exhaled breath-H2S analysis by the combined use of moisture trapping, cryogenic pre-concentration, thermal desorption and GC technique, we quantitatively evaluated the moisture removing and H2S permeating efficiency of CaCl2, silica gel, molecular Sieve 4A, a membrane type dryer, and sample cooling (in the range of 45oC to -20oC) using Selected Ion Flow Tube – Mass Spectrometry (SIFT-MS) as a simultaneous means of detecting H2O and H2S in human breath samples spiked with H2S and humidified with distilled water. While CaCl2, membrane type dryer, and sample freezing remove moisture to as low as 0.4% without affecting H2S, silica gel and molecular Sieve 4A removed both moisture and H2S in the studied samples. Cartridges containing 0.4 g – 1.6 g CaCl2 as desiccant appear to be a viable alternative to more expensive membrane dryers.

In this study, transition metals (Cu(II), Co(II), Ni(II), Fe(III) and Ru(III)), complexes of Schiff base derived from (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-carboxypropan-2-yloxyimino)acetamido)-8-oxo-3-(pyridinium— ylmethyl)-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate, Ceftazidime; CFZ) and p-dimethylaminobenzaldehyde (DAB) were prepared and characterized by the analytical and spectroscopic methods. The analytical data show the composition of the metal complex to be [M(CFZ-DAB)Cln]Cl, where CFZ-DAB is the Schiff base ligand and n=1 for Cu(II), Co(II), Ni(II) and n=2 for Fe(III) and Ru(III) complexes. The conductance data indicate that all the complexes are strongelectrolytes. The compound (CFZ-DAB) behaves as a tridentate ligand. But, obtained complexes have mononuclear nature. The electrochemical properties of the metal complexes were investigated by cyclic voltammetry (CV) using glassy carbon electrode. The oxidation/reduction of metal complexes wasirreversible/reversible and exhibited diffusion controlled process depending on pH. The dependence of intensities of currents and potentials on pH, concentration, scan rate, nature of the buffer was investigated. The oxidation/reduction mechanism was proposed and discussed.