Fully automated sample treatment method for high throughput proteome analysis

The bottom-up strategy for proteome analysis typically employs a multistep sample preparation workflow that suffers from being time-consuming and sample loss or contamination caused by the off-line manual operation.Herein,we developed a hollow fibre membrane(HFM)-aided fully automated sample treatment(FAST)method.Due to the confinement effects of HFMs and the immobilized enzymatic reactor,the proteome samples could be denatured,reduced,desalted and digested within 8–20 min via the one-stop service.This method also showed superiority in trace sample analysis.In one and half hours,we could identify about 1,600 protein groups for 500 HeLa cells as the starting materials,1.5–8 times more than those obtained by previously reported methods.Through the on-line combination of FAST with nano-liquid chromatography-electrospray ionization tandem mass spectrometry(nanoLC-ESI-MS/MS),we further established a fully integrated platform for label-free quantification of proteome with high reproducibility and precision.Collectively,FAST presented here represents a major advance in the high throughput sample treatment and quantitative analysis of proteomes.     

Fully automated liquid chromatography-mass spectrometry determination of 17beta-estradiol in river water

Surface modified molecularly imprinted polymers (SM-MIPs) for 17beta-estradiol (E2), utilizing 6-ketoecradiol as a pseudo template were prepared. MIPs for E2 were synthesized using 4-vinyl pyridine and ethylene dimethacrylate as a functional monomer and cross-linking agent, respectively. MIPs selectively retained E2 and provided excellent chromatographic resolution from interfering compounds inherent in river water sample matrices. Therefore, freshly prepared MIPs were applied to quantitative mass spectrometric (negative electrospray ionization mode) detection of low levels of E2 in river water sample. In order to pre-concentrate the target compound for HPLC analysis, column switching was coupled with a pretreatment column packed with the MIPs. The repeatability of actual determinations of river water sample, in which background E2 was not detected, spiked with 50 ng/L of E2 was 2.2% RSD with a detection limit and qualification limit of 1.8 and 5.4 ng/L, respectively. Surface modification of MIP particlefs packed in the pretreatment column provided selective affinity and on-line concentration of low levels of E2 while simultaneously eliminating sample matrix interference, resulting in a significant increase in sensitivity and reproducibility for liquid chromatography-mass spectrometry analysis of E2 in river water sample.     

Fully automated and computer-controlled polarography

Summary In previous papers a fully automated and computer-controlled polarographic equipment [6] and its precision and accuracy [5] was reported. Automated development of methods can be done by computer-controlled dosage of the substance to be determined to various buffers. From 3D-plots optimum conditions can be easily seen. Calibration is done by computer-controlled dosage of the substance to be determined to different electrolyte solutions, repeated measurements and repeated dosage. Change of samples and transfer to degassing vessel and measuring cell are controlled by the computer.     

Electrochemical biosensors for food analysis

Abstract  Food analysis has become a very important and interesting area of research because of the rapid expansion of food trade and highly increased mobility of today’s populations. Food quality control is essential both for consumer protection and also for the food industry. Application of the electrochemical biosensors in the field of food analysis is promising. This review covers the recent developments and issues in electrochemical biosensors for food analysis, such as ease of preparation, robustness, sensitivity, and realization of mass production of the detection strategies. This review also emphasizes the current development of electrochemical biosensors combined with nanotechnology. Graphical abstract        

Electrochemical biosensors for food analysis

Abstract  

Food analysis has become a very important and interesting area of research because of the rapid expansion of food trade and highly increased mobility of today’s populations. Food quality control is essential both for consumer protection and also for the food industry. Application of the electrochemical biosensors in the field of food analysis is promising. This review covers the recent developments and issues in electrochemical biosensors for food analysis, such as ease of preparation, robustness, sensitivity, and realization of mass production of the detection strategies. This review also emphasizes the current development of electrochemical biosensors combined with nanotechnology.     

Fully automated high-performance liquid chromatographic assay for the analysis of free catecholamines in urine

A totally automated and reliable high-performance liquid chromatographic method is described for the routine determination of free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines were isolated from urine samples using small alumina columns. A standard automated method for pH adjustment of urine before the extraction step has been developed. The extraction was performed on an ASPEC (Automatic Sample Preparation with Extraction Columns, Gilson). The eluate was collected in a separate tube and then automatically injected into the chromatographic column. The catecholamines were separated by reversed-phase ion-pair liquid chromatography and quantified by fluorescence detection. No manual intervention was required during the extraction and separation procedure. One sample may be run every 15 min, ca. 96 samples in 24 h. Analytical recoveries for all three catecholamines are 63-87%, and the detection limits are 0.01, 0.01, and 0.03 microM for norepinephrine, epinephrine and dopamine, respectively, which is highly satisfactory for urine. Day-to-day coefficients of variation were less than 10%.     

Cell-based electrochemical biosensors for water quality assessment

During recent decades, extensive industrialisation and farming associated with improper waste management policies have led to the release of a wide range of toxic compounds into aquatic ecosystems, causing a rapid decrease of world freshwater resources and thus requiring urgent implementation of suitable legislation to define water remediation and protection strategies. In Europe, the Water Framework Directive aims to restore good qualitative and quantitative status to all water bodies by 2015. To achieve that, extensive monitoring programmes will be required, calling for rapid, reliable and cost-effective analytical methods for monitoring and toxicological impact assessment of water pollutants. In this context, whole cell biosensors appear as excellent alternatives to or techniques complementary to conventional chemical methods. Cells are easy to cultivate and manipulate, host many enzymes able to catalyse a wide range of biological reactions and can be coupled to various types of transducers. In addition, they are able to provide information about the bioavailability and the toxicity of the pollutants towards eukaryotic or prokaryotic cells. In this article, we present an overview of the use of whole cells, mainly bacteria, yeasts and algae, as sensing elements in electrochemical biosensors with respect to their practical applications in water quality monitoring, with particular emphasis on new trends and future perspectives. In contrast to optical detection, electrochemical transduction is not sensitive to light, can be used for analysis of turbid samples and does not require labelling. In some cases, it is also possible to achieve higher selectivities, even without cell modification, by operating at specific potentials where interferences are limited.     

Fully automated capillary isotachophoresis of proteins

An apparatus for fully automated capillary isotachophoresis was constructed. A commercial apparatus (Shimadzu IP-2A) was modified in the electrolyte pumping system and the flow lines were simplified. An automatic sampler was used for sequential sampling. The equipment, pumps, the sampler, a high-voltage DC power supply, and a recorder, were controlled by a system controller which comprises a microcomputer and a BASIC program for time-control of the equipments. The apparatus was successfully used for the automated sequential analysis of human serum proteins. Forty serum samples were analyzed within 17 h without manual operation and for each sample the serum proteins were resolved into about twenty UV peaks or shoulders.     

Fully automated synthesis of N-labeled nitrosothiols

In the present Letter, a fast, automated, and reproducible method for the synthesis of S-[¹³N]nitrosothiols is reported. The labeling strategy is based on trapping in an anion exchange resin. The reaction with thiols in acidic media led to the formation of the desired nitrosothiols in short reaction times (60 s) with excellent radiochemical conversions (from 48.7% to 74.5%). Final radiotracers were purified by HPLC. Good radiochemical yields (from 33.8% to 60.6%, decay corrected) and radiochemical purities (>99%) were obtained in all cases. Stability of the labeled compounds was checked.     

Fully automated online multi-dimensional protein profiling system for complex mixtures

For high throughput proteome analysis of highly complex protein mixtures, we have constructed a fully automated online system for multi-dimensional protein profiling, which utilizes a combination of two-dimensional liquid chromatography and tandem mass spectrometry (2D-LC-MS-MS), based on our well-established offline system described previously [K. Fujii, T. Nakano, T. Kawamura, F. Usui, Y. Bando, R. Wang, T. Nishimura, J. Proteome Res. 3 (2004) 712]. A two-valve switching system on a programmable auto sample injector is utilized for online two-dimensional chromatography with strong cation-exchange (SCX) and reversed-phase (RP) separations. The SCX separation is carried out during the equilibration of RP chromatography and the entire sequence of analysis was performed under fully automated conditions within 4 h, based on six SCX fractionations, and 40 min running time for the two-dimensional RP chromatography. In order to evaluate its performance in the detection and identification of proteins, digests of six standard proteins and yeast 20S proteasome have been analyzed and their results were compared to those obtained by the one-dimensional reversed-phase chromatography system (ID-LC-MS-MS). The 2D-LC-MS-MS system demonstrated that both the number of peptide fragments detected and the protein coverage had more than doubled. Furthermore, this multi-dimensional protein profiling system was also applied to the human 26S proteasome, which is one of the highly complex protein mixtures. Consequently, 723 peptide fragments were identified as 31 proteasome components, together with other coexisting proteins in the sample. The identification could be comprehensively performed with a 63% sequence coverage on an average, and additionally, with modifications at the N-terminus. These results indicated that the online 2D-LC-MS-MS system being described here is capable of analyzing highly complex protein mixtures in a high throughput manner, and that it would be applicable to dynamic proteomics.     

Fully automated sample handling system for liquid chromatography based on pre-column technology and automated cartridge exchange

The design of an automated cartridge exchange module for on-line sample handling in liquid chromatography is described. When combined with a low-cost purge pump, a solvent selection valve and an auto-sampler, a fully automated sample handling system is obtained. Samples are sorbed on a disposable cartridge packed with 40 micron octyl-bonded silica, purged for clean-up and eluted on-line to the analytical column. Unattended operation of the system is demonstrated for various examples, i.e., the determination of anti-epileptic drugs in serum, an anti-cancer drug in plasma, barbiturates in urine, phenylurea herbicides in river water and caffeine in a soft drink.     

Fully automated polymer-assisted synthesis of 1,5-biaryl pyrazoles

The polymer-assisted solution-phase (PASP) synthesis of a 192-member 2-D array of 1,5-biaryl pyrazoles 4[1-12,1-16] is reported. The synthesis was performed in a fully automated manner using a multiprobe top-filtration robot and incorporates a "catch and release" step to afford library compounds directly in high yield and purity.     

Compact amperometric algal biosensors for the evaluation of water toxicity

Unicellular microalga Chlorella vulgaris was entrapped in an alginate gel or a polyion complex membrane immobilized directly on the surface of a transparent indium tin oxide electrode. Photosynthetically generated oxygen of the immobilized algae was monitored amperometically. Responses of the algal biosensor to four toxic compounds, 6-chloro-N-ethyl-N-isopropyl-1,3,5-triazine-2,4-diamine (atrazine), 3-(3,4-dichlorophenyl)-1,1-diethylurea (DCMU), toluene and benzene, were evaluated as inhibition ratios of the reduction current. The concentrations that give 50% inhibition of the oxygen reduction current (IC^′₅₀) for atrazine, DCMU, toluene and benzene were 2.0, 0.05, 1550 and 3000 μmol dm⁻³, respectively. There was a good correlation between these data and those of the conventional standard growth test. In comparison with the conventional algal biosensors based on the Clark-type oxygen electrode, the present sensor is much smaller and less expensive, and its assay time is much shorter (≤200 s).     

Recent developments in potentiometric biosensors for biomedical analysis

A large variety of potentiometric biosensors is developed using biocatalytic and bioaffinity-based biosensing schemes. However, only few of them could be applied for the biomedical analysis. The most promising are those for the detection of main products of protein metabolism, namely urea and creatinine. A novel group of potentiometric biosensors is constituted by bioaffinity-based devices that could be used for immunoassays or genoanalysis. This paper reviews the recent trends in these fields as well as discusses advantages, limitations and pitfalls of the developed biosensors. Some potentiometric biosensors useful for real biomedical analysis are reported in detail.     

Fully automated in-tube solid-phase microextraction for liquid samples coupled to gas chromatography

An online device is described in which analytes are extracted from a liquid sample by means of in-tube solid-phase microextraction (in-tube SPME), pulse released by rapid heating, and transferred to a gas chromatograph in a fully automated way. Switching of the sample and gas flows as well as the heating of the extraction tube and the valves is controlled by a remote computer system. Results obtained for river water and for aqueous standard solutions of phenanthrene are presented and are compared to the performance of standard SPME.     

Fully automated standard addition method for the quantification of 29 polar pesticide metabolites in different water bodies using LC-MS/MS

A reliable quantification by LC-ESI-MS/MS as the most suitable analytical method for polar substances in the aquatic environment is usually hampered by matrix effects from co-eluting compounds, which are unavoidably present in environmental samples. The standard addition method (SAM) is the most appropriate method to compensate matrix effects. However, when performed manually, this method is too labour- and time-intensive for routine analysis. In the present work, a fully automated SAM using a multi-purpose sample manager “Open Architecture UPLC®-MS/MS” (ultra-performance liquid chromatography tandem mass spectrometry) was developed for the sensitive and reliable determination of 29 polar pesticide metabolites in environmental samples. A four-point SAM was conducted parallel to direct-injection UPLC-ESI-MS/MS determination that was followed by a work flow to calculate the analyte concentrations including monitoring of required quality criteria. Several parameters regarding the SAM, chromatography and mass spectrometry conditions were optimised in order to obtain a fast as well as reliable analytical method. The matrix effects were examined by comparison of the SAM with an external calibration method. The accuracy of the SAM was investigated by recovery tests in samples of different catchment areas. The method detection limit was estimated to be between 1 and 10 ng/L for all metabolites by direct injection of a 10-μL sample. The relative standard deviation values were between 2 and 10 % at the end of calibration range (30 ng/L). About 200 samples from different water bodies were examined with this method in the Rhine and Ruhr region of North Rhine-Westphalia (Germany). Approximately 94 % of the analysed samples contained measurable amounts of metabolites. For most metabolites, low concentrations ≤0.10 μg/L were determined. Only for three metabolites were the concentrations in ground water significantly higher (up to 20 μg/L). In none of the examined drinking water samples were the health-related indication values (between 1 and 3 μg/L) for non-relevant metabolites exceeded.     

Fully automated sequential solid phase approach towards viral RNA-nucleopeptides

The synthesis of two ribonucleoprotein fragments of unprecedented complexity is reported. These hybrid biomolecules are prepared combining the use of an automated solid phase peptide and oligonucleotide synthesizer on a single solid support.     

Fully automated continuous flow synthesis of 4,5-disubstituted oxazoles

[Structure: see text] A multipurpose mesofluidic flow reactor capable of producing gram quantities of material has been developed as an automated synthesis platform for the rapid on-demand synthesis of key building blocks and small exploratory libraries. The reactor is configured to provide the maximum flexibility for screening of reaction parameters that incorporate on-chip mixing and columns of solid supported reagents to expedite the chemical syntheses.