Application of a temperature-controllable microreactor to simple and rapid protein identification using MALDI-TOF MS

We have carried out a simultaneous thermal denaturation and trypsin digestion of proteins using a temperature-controllable microreactor. This is a simple and rapid sample preparation technique for use before matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In contrast to a conventional sample preparation method, which involves several chemical treatments, our sample preparation was performed using only trypsin digestion with the thermal denaturation of the target protein. Optimization of the reactor operational parameters for trypsin digestion using a temperature-controllable microreactor was carried out. The entire trypsin digestion procedure took about 11 min, and consisted of 1 min for the thermal denaturation of the sample protein (3 microl, 0.2 microM) at 85 degrees C, and 10 min for digestion of the protein at 37 degrees C. The resulting sequence coverage ranged from 24% to 57%, which was sufficient for practical protein identification.     

Microtiter plate based chemistry and in situ screening: a useful approach for rapid inhibitor discovery

The use of libraries extracted from nature or constructed by combinatorial chemistry, have been widely appreciated in the drug discovery area. In this perspective, we present our contribution to the field of enzyme inhibitor discovery using a useful approach that allows diversification of a common core in a microtiter plate followed by in situ screening. Our method relies on an organic reaction that is highly selective, high yielding, amenable to the microscale and preferably can be performed in water. The core can be a designed molecule based on the structural and mechanistic information of the target, a compound with a weak binding affinity, or a natural product. Several reactions were found useful for this approach and were applied to the rapid discovery of potent inhibitors of representative enzymes.     

SpectraBank: an open access tool for rapid microbial identification by MALDI-TOF MS fingerprinting

MALDI-TOF MS has proved to be an accurate, rapid, and cost-effective technique for microbial identification in which the spectral fingerprint of an unknown strain can be compared to a database of spectra from reference strains. Most of the existing databases are private and often costly to access, and little spectral information is shared among researchers. The objective of the present communication is to introduce the SpectraBank database (http://www.spectrabank.org), which provides open access MALDI-TOF mass spectra from a variety of microorganisms. This work aims to familiarize readers with the SpectraBank database, from the sample preparation, data collection, and data analysis to how the spectral reference data can be used for microbial species identification. The database currently includes more than 200 MALDI-TOF MS spectra from more than 70 bacterial species and links to the freely available web-based application SPECLUST (http://bioinfo.thep.lu.se/speclust.html) to allow comparisons of the obtained peak mass lists and evaluate phyloproteomic relationships. The SpectraBank database is intended to be expanded by the addition of new spectra from microbial strains, obtained in our laboratory and by other researchers.     

A sensitive colorimetric label-free assay for trypsin and inhibitor screening with gold nanoparticles

Gold nanoparticles (Au-NPs) with negative charges aggregate in the presence of Arg(6) due to electrostatic interactions resulting in the red-shift of the plasmon absorption. But, after incubation of Arg(6) with trypsin the aggregation of Au-NPs can be prohibited. Accordingly, a newly designed-Au-NPs based colorimetric assay method for trypsin activity is established. Trypsin with a concentration as low as 1.6 ng mL(-1) can be assayed with this new colorimetric assay. This colorimetric label-free assay for trypsin can be performed in aqueous solution and both Au-NPs and Arg(6) are easily accessible. Thus, this assay method is useful for screening inhibitors of trypsin.     

Solid-state NMR spectroscopy as a tool supporting optimization of MALDI-TOF MS analysis of polylactides

We report systematic structural studies of poly(l-lactide) (PLLA) employing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and solid-state NMR spectroscopy. (13)C cross polarization magic angle spinning (CP/MAS) NMR data for 1,8-dihydroxy-9-anthracenone (DT), 2,5-dihydroxybenzoic acid (DHB), 2-(4-hydroxyphenylazo)-benzoic acid (HABA), and trans-3-indoleacrylic acid (IAA), four matrices commonly used in MALDI-TOF analysis of polymers, were analyzed to test the influence of crystallization conditions (solvent, inorganic salt) on sample morphology. (13)C principal elements of chemical shift tensor (CST) and line-shape analyses were employed to study of the nature of hydrogen bonding and to evaluate the crystallinity and amorphicity of the pure polymer. NMR parameters for PLLA were compared with data for polylactide crystallized with the four matrices under different conditions with the addition of two inorganic salts as cationization agents. This study revealed that the semicrystalline structure of the polymer does not change when it is embedded in the matrix.     

Biorecognition-modulated ion fluxes through functionalized gold nanotubules as a novel label-free biosensing approach

A novel biosensing principle is presented, based on the potentiometric monitoring of an indicator ion such as Ca2+, whose zero-current flux through chemically modified nanochannels is altered by biorecognition events.     

Porous anodic alumina membrane as a sample support for MALDI-TOF MS analysis of salt-containing proteins

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF MS) analysis of proteins in salt-containing solution was performed for the first time using porous anodic alumina (PAA) membrane as sample support. The resulting spectral quality of proteins under standard sample preparation conditions was superior to that of normal metal sample stages. Analysis of phosphate-doped protein solutions indicated that porous anodic alumina membranes as a target yielded better results than a metallic target for salt-containing solutions. Because of the biocompatibility of the PAA, proteins can be adsorbed on the PAA and thus a washing process can be introduced to remove the salts from the PAA target before MS analysis. This desalting step significantly enhanced spectral quality, and better signal-to-noise ratios were obtained. The present technique is promising for proteomics research.     

A cyclodextrin host-guest recognition approach to a label-free electrochemical DNA hybridization biosensor

A novel label-free electrochemical DNA hybridization biosensor using a β-cyclodextrin/poly(N-acetylaniline)/carbon nanotube composite modified screen printed electrode (CD/PNAANI/CNT/SPE) has been developed. The proposed DNA hybridization biosensor relies on the intrinsic oxidation signals of guanine (G) and adenine (A) from single-stranded DNA entered into the cyclodextrin (CD) cavity. Due to the binding of G and A bases to complementary cytosine and thymine bases in dsDNA, the signals obtained for ssDNA were much higher than that of dsDNA. The synergistic effect of the multi-walled carbon nanotubes provides a significantly enhanced voltammetric signal, and the CD encapsulation effect makes anodic peaks of G and A shift to less positive potentials than that at the bare SPE. The peak heights of G and A signals are dependent on both the number of the respective bases in oligonucleotides and the concentration of the target DNA sequences. Hybridization of complementary strands was monitored through the measurements of oxidation signal of purine bases, which enabled the detection of target sequences from 0.01 to 1.02 nmol μl(-1) with the detection limit of target DNA as low as 5.0 pmol μl(-1) (S/N = 3). Implementation of label-free and homogeneous electrochemical hybridization detection constitutes an important step toward low-cost, simple, highly sensitive and accurate DNA assay. Discrimination between complementary, noncomplementary, and two-base mismatch targets was easily accomplished using the proposed electrode.     

Development of an LC-MS/MS method for aromatase inhibitor screening

Aromatase (CYP 19A1) is a key steroidogenic enzyme that catalyzes the conversion of androgen to estrogen. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for aromatase inhibitor screening was developed and validated. The substrate androstenedione was incubated with human CYP 19A1 supersomes in the presence of NADPH for 30 min, and estrone formation was determined by LC-MS/MS analysis. Cortisone was used as internal standard. The incubation mixture was extracted using a liquid-liquid extraction method with ethyl acetate. Chromatographic separation was achieved using a C₁₈ column (3.0?×?50 mm, 2.7 μm) with a mobile phase consisting of 0.1 % formic acid/acetonitrile adopting gradient elution at a flow rate of 0.4 mL/min. The mass spectrometer was operated in positive electrospray ionization mode. The precursor-product ion pairs used for multiple reaction monitoring were m/z 287→97 (androstenedione), m/z 271?→?159 (estrone), and m/z 361?→?163 (IS, cortisone). The developed method met the required criteria for the validation of bioanalytical methods. The validated method was successfully applied to evaluate aromatase inhibitory activity of plants extracts of Simaroubaceae.

Efficient analysis of non-polar environmental contaminants by MALDI-TOF MS with graphene as matrix

In this Application Note, we describe, for the first time, the rapid analysis of hydrophobic compounds present in environmental contaminants, which includes polycyclic aromatic hydrocarbons (PAHs) and estrogen, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the use of graphene as matrix. MALDI-TOF MS with conventional matrix has limitations in analyzing low-polarity compounds owing to their difficulty in ionization. We demonstrate that compared with conventional matrix, graphene displays higher desorption/ionization efficiencies for PAHs, and no fragment ions are observed. The method also holds potential in quantitative analysis. In addition, the ionization signal increases with the increasing number of benzene rings in the PAHs, suggesting that graphene binds to PAHs via π–π stacking interactions. Furthermore, graphene as adsorbent for solid-phase extraction of coronene from river water sample displays good performance with a detection limit of 10^–7 M. This work provides a novel and convenient method for analyzing low-polarity environmental contaminants by MALDI-TOF MS.     

Back to basics: label-free technologies for small molecule screening

Small molecule high-throughput screening in drug discovery today is dominated by techniques which are dependent upon artificial labels or reporter systems. While effective, these approaches can be affected by certain experimental limitations, such as conformational restrictions imposed by the selected label or compound fluorescence/quenching. Label-free approaches potentially address many of these issues by allowing researchers to investigate more native systems without fluorescence- or luminescence-based readouts. However, due to throughput and expense constraints, label-free methods have been largely relegated to a supporting role as the basis of secondary assays. In this review, we describe recent improvements in impedance-based, optical biosensor-based, automated patch clamp and mass spectrometry technologies that have enhanced their ease of use and throughput and, hence, their utility for primary screening of small- to medium-sized compound libraries. The ultimate maturation of these techniques will enable drug discovery researchers to screen large chemical libraries against minimally manipulated biological systems.     

Mesoporous Fe2O3 microspheres: rapid and effective enrichment of phosphopeptides for MALDI-TOF MS analysis

Mesoporous Fe(2)O(3) microspheres have been successfully synthesized by the polymerization (urea and formaldehyde)-induced ferric hydroxide colloid aggregation. The urea-formaldehyde resin was removed by calcination in air. The obtained mesoporous Fe(2)O(3) materials have spherical morphology with uniform particle size of approximately 3.0 microm and porous surface with large inter-particle pores of approximately 48.0 nm. The surface area is as large as approximately 33.3 m(2)/g and the pore volume is 0.31 cm(3)/g. The mesoporous Fe(2)O(3) microspheres were used for the enrichment of phosphopeptides for the first time, in which high sensitivity, selectivity and capacity of specifically enriched phosphopeptides were achieved under a mild condition in a relative short time. After enriched from tryptic digest products of beta-casein by the novel mesoporous Fe(2)O(3) microspheres, phosphopeptides can be selectively detected with high intensity in MALDI-TOF mass spectrometry. Elimination of "shadow effect" was observed by using mesoporous Fe(2)O(3) microspheres, and the detectable limitation is 5x10(-10) M. This material is also effective for enrichment of phosphopeptides from the complex tryptic digests of commercial phosphoprotein casein, with much more phosphorylated sites (26 in 27 of total) and higher signal/noise ratio in the MALDI-TOF mass spectrometry, compared to commercial Fe(2)O(3) nanoparticles. It shows a great potential application in the field of rapid and effective isolation of phosphopeptides.     

A parallel approach to post source decay MALDI-TOF analysis

We present a novel enhancement to matrix-assisted laser desorption ionization (MALDI) post-source decay (PSD) analysis whereby fragment ions from multiple precursor ions are acquired into the same spectrum without employing a timed ion gate to preselect each parent ion. Fragment ions are matched to their corresponding precursor ions by comparing spectra acquired at slightly different reflectron electric fields. By measuring the difference in time-of-flight (TOF) between the two spectra for each fragment, it is possible to calculate the mass of the fragment ion and its parent. This new "parallel PSD" technique reduces analysis time and consumes less sample than conventional PSD, which requires an ion gate for serial preselection of precursor ions.     

Using oxidized carbon nanotubes as matrix for analysis of small molecules by MALDI-TOF MS

Oxidized carbon nanotubes are tested as a matrix for analysis of small molecules by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Compared with nonoxidized carbon nanotubes, oxidized carbon nanotubes facilitate sample preparation because of their higher solubility in water. The matrix layer of oxidized carbon nanotubes is much more homogeneous and compact than that of nonoxidized carbon nanotubes. The efficiency of desorption/ionization for analytes and the reproducibility of peak intensities within and between sample spots are greatly enhanced on the surface of oxidized carbon nanotubes. The advantage of the oxidized carbon nanotubes in comparison with alpha-cyano-4-hydroxycinnamic acid (CCA) and carbon nanotubes is demonstrated by MALDI-TOF-MS analysis of an amino acid mixture. The matrix is successfully used for analysis of synthetic hydroxypropyl beta-cyclodextrin, suggesting a great potential for monitoring reactions and for product quality control. Reliable quantitative analysis of jatrorrhizine and palmatine with a wide linear range (1-100 ng/mL) and good reproducibility of relative peak areas (RSD less than 10%) is achieved using this matrix. Concentrations of jatrorrhizine (8.65 mg/mL) and palmatine (10.4 mg/mL) in an extract of Coptis chinensis Franch are determined simultaneously using the matrix and a standard addition method.     

Using MALDI-TOF MS coupled with a high-mass detector to directly analyze intact proteins in thyroid tissues

Protein analysis is vital for biological and clinical research, but the measurement of unseparated, intact and high-mass proteins is also a challenging task by mass spectrometry-based methods. Here, we present a protocol for rapid and high-throughput analysis of intact proteins in tissue samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDITOF MS) combined with a high-mass detector platform. The method involves tissue specimens that undergo a simple protein extraction before MALDI-MS analysis. Using this method, the high abundance proteins in human thyroid carcinoma and paracarcinoma tissues were successfully investigated, and the mass spectra of the tissues of the 30 illustrated thyroid cancers showed remarkable differences. The peak intensity revealed a significant increase in human albumin in thyroid carcinoma tissues(p0.05). To validate the feasibility and credibility of this method, label-free proteomics quantitative analysis and Western blotting were used to relatively quantify the proteins in these tissues. Those results demonstrated a nearly 3-fold difference in human albumin levels between thyroid carcinoma and para-carcinoma tissues, which were consistent with the results of our method. The advantages of our method are easy sample handling, remarkable reproducibility and the ability to analyze high-mass proteins without digestion, which make them have the potential to be used in biological research and in clinical practice.     

Potential of MALDI-TOF mass spectrometry as a rapid detection technique in plant pathology: identification of plant-associated microorganisms

Plant diseases caused by plant pathogens substantially reduce crop production every year, resulting in massive economic losses throughout the world. Accurate detection and identification of plant pathogens is fundamental to plant pathogen diagnostics and, thus, plant disease management. Diagnostics and disease-management strategies require techniques to enable simultaneous detection and quantification of a wide range of pathogenic and non-pathogenic microorganisms. Over the past decade, rapid development of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for characterization of microorganisms has enabled substantially improved detection and identification of microorganisms. In the biological sciences, MALDI-TOF MS is used to analyze specific peptides or proteins directly desorbed from intact bacteria, fungal spores, nematodes, and other microorganisms. The ability to record biomarker ions, in a broad m/z range, which are unique to and representative of individual microorganisms, forms the basis of taxonomic identification of microorganisms by MALDI-TOF MS. Recent advances in mass spectrometry have initiated new research, i.e. analysis of more complex microbial communities. Such studies are just beginning but have great potential for elucidation not only of the interactions between microorganisms and their host plants but also those among different microbial taxa living in association with plants. There has been a recent effort by the mass spectrometry community to make data from large scale mass spectrometry experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MALDI-TOF MS as a universal technique for detection of plant pathogens and non-pathogens. The effects of experimental conditions are sufficiently understood, reproducible spectra can be obtained from computational database search, and microorganisms can be rapidly characterized by genus, species, or strain.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

Supramolecular displacement-mediated activation of a silent fluorescence probe for label-free ligand screening

We report a new approach for the rapid screening of analyte binding affinities for a target protein. We demonstrate that a molecular probe, with a pro-fluorophore substrate and ligand moieties, can be hindered from enzymatic access when bound to the target protein. When analytes displace the probe from the protein's binding pocket, a fluorescence profile is generated. This profile is used to discriminate analytes based on their relative binding affinities.     

基于MALDI-TOF MS微生物检测的一次性纸基靶板开发

针对基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)目前使用的不锈钢靶板或一次性可抛弃塑料靶板存在的清洗流程复杂,易有残留从而产生交叉污染或需要回收、污染环境等问题,开发了一种可一次性使用的纸基靶板。该靶板成本低,使用后方便处理,不会对环境造成污染。并利用标准品从分辨率、灵敏度、重现性、微生物鉴定4个方面对纸基靶板进行了测试,结果表明开发的纸基靶板在分辨率、灵敏度等指标方面与钢靶板基本一致,样品检测重现性较好,具有应用于临床微生物检测的潜力。