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PubMed2006

Methods for samples preparation in proteomic research.

Sample preparation is one of the most crucial processes in proteomics research. The results of the experiment depend on the condition of the starting material. Therefore, the proper experimental model and careful sample preparation is vital to obtain significant and trustworthy results, particularly in comparative proteomics, where we are usually looking for minor differences between experimental-, and control samples. In this review we discuss problems associated with general strategies of samples preparation, and experimental demands for these processes.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Sample preparation: quo vadis?

The sample preparation step in an analytical process typically consists of an extraction procedure that results in the isolation and enrichment of components of interest from a sample matrix. Extraction can vary in degree of selectivity, speed, and convenience and depends not only on the approach and conditions used but on the geometric configurations of the extraction phase. Increased interest in sample preparation research has been generated by the introduction of nontraditional extraction technologies. These technologies address the need for reduction of solvent use, automation, and miniaturization and ultimately lead to on-site in situ and in vivo implementation. These extraction approaches are frequently easier to operate but provide optimization challenges. More fundamental knowledge is required by an analytical chemist not only about equilibrium conditions but, more importantly, about the kinetics of mass transfer in the extraction systems. Optimization of this extraction process enhances overall analysis. Proper design of the extraction devices and procedures facilitates convenient on-site implementation, integration with sampling, and separation/quantification, automation, or both. The key to rational choice, optimization, and design is an understanding of the fundamental principles governing mass transfer of analytes in multiphase systems. The objective of this perspective is to summarize the fundamental aspects of sample preparation and anticipate future developments and research needs.

Journal Article↗
PubMed1989

Sample preparation for biomedical analysis.

A review of sample preparation techniques for biomedical analysis is presented. Firstly, sample preparation techniques can be divided into unit operations which can be classified into four groups: release of the analyte from the matrix, removal of endogenous material, liquid handling procedures and the enhancement of selectivity and sensitivity. The concept of unit operations gives an analyst a tool with which to evaluate critically any method for preparing a sample for analysis. Secondly, the major techniques of sample preparation (protein precipitation, liquid-liquid extraction, liquid-solid extraction and high-performance liquid chromatography, HPLC) are discussed and their advantages and disadvantages presented. Thirdly, the rationale for the automation of sample preparation is reviewed; in general liquid-solid extraction and HPLC are the best techniques for automation. The means by which this can be effected (either flexible or dedicated automation) is discussed. Finally, techniques such as supercritical fluid extraction, micellar liquid chromatography, microwave energy and immunoextraction which may be applied to biomedical sample preparation are evaluated briefly.

Chemistry Techniques, Analytical↗
PubMed2003

Miniaturized sample preparation combined with liquid phase separations.

Miniaturized sample preparation methods designed as the sample pretreatment for liquid phase separations, such as liquid chromatography, capillary electrophoresis and capillary electrochromatography, have been reviewed especially for the on-line coupling of the sample preparation process and the separation process. The development of the desorption interfaces for the effective combining of the sample preparation and subsequent liquid phase separations is briefly described along with the applications of the combined analytical systems to the analysis of complex sample mixtures such as biological and environmental matrices. Novel use of fine polymeric filaments as the extraction medium for microscale liquid phase separation methods are investigated and a comparison is made with other sample preparation techniques. Polymer coating onto the fibrous material is also introduced to further develop microscale sample preparation methods with improved extraction performance. Several other microscale sample preparation methods having a potential compatibility to the liquid phase separations are also described for future applications of these techniques.

Chromatography, Liquid↗
PubMed2002

Effect of sample preparation and pH-mediated sample stacking on the analysis of multiplexed short tandem repeats by capillary electrophoresis.

An amplification system was used in this study to evaluate the effect of sample preparation and its injection on the electrophoretic separation by capillary electrophoresis using a commercially available capillary electrophoresis instrument. The effect of dilution of amplified samples in water and in different sources of formamide (with conductivity values ranging from 47 to 1000 microS) was evaluated, as was contamination of the sample with high DNA concentrations or buffer salts. Although resolution remained constant in the different solvents tested, the sensitivity increased in samples diluted in water and high-purity formamide. An on-column sample preconcentration method for capillary-based DNA analysis was evaluated to increase the sensitivity of low-quality samples. This technique, pH-mediated sample stacking, is based on the injection of NaOH immediately before sample injection: A neutralization reaction occurs between OH- and tris+ ions so that a low-conductivity zone is created at the head of the capillary. DNA fragments are concentrated at the front of this zone. Using coated capillaries with hydroxycellulose 2% (MW 250,000) as a separation matrix, an improvement in sensitivity was detected in all the solvents studied. The gain in sensitivity was higher for more conductive solvents, and was not correlated with the size of the DNA fragments.

Electrophoresis, Capillary↗
PubMed2002

Sorptive sample preparation -- a review.

Most sample-enrichment procedures currently available rely on adsorption of the analytes of interest by a suitable adsorbent material. Although good performance can be obtained for many practical problems, in some cases the applicability of adsorptive sample preparation falls short, particularly for the enrichment of polar and/or high-molecular-weight compounds, especially in combination with thermal desorption. Because of the very strong retention of adsorbent materials, undesired effects such as incomplete desorption and artifact formation are observed. Polar solutes are easily adsorbed but readily undergo surface-catalyzed reactions and on desorption yield compounds different than those originally sampled. High-molecular-weight compounds cannot be desorbed because of extremely strong interactions with the adsorbent and their low volatility. To overcome some of these problems sample-preparation techniques based on polydimethylsiloxane sorption have been developed over the past 15 years. In contrast with adsorptive trapping, sorption is based on dissolution of the analytes in a liquid polymeric material. This is a much more inert means of solute retention which overcomes some of the limitations encountered when working with adsorbents. In this contribution, the basic principles of sorption, the different instrumentation used, and applications of the technique will be reviewed. The review covers the sorptive sample-preparation techniques, open-tubular trapping (OTT), solid-phase microextraction (SPME), gum-phase extraction (GPE), equilibrium gum-phase extraction (EGPE), and stir-bar-sorptive extraction (SBSE). Because of the nature of sorptive sample-preparation techniques, which perform particularly well in combination with thermal desorption, this review focuses strongly on gas chromatography as the means of chemical analysis.

Chromatography, Gas↗
PubMed2006

A simple supported liquid hollow fiber membrane microextraction for sample preparation of trihalomethanes in water samples.

A simple and efficient liquid-phase microextraction (LPME) technique using a supported liquid hollow fiber membrane, in conjunction with gas chromatography-electron capture detector has been developed for extraction and determination of trihalomethanes (THMs) in water samples. THMs were extracted from water samples through an organic extracting solvent impregnated in the pores and filled inside the porous hollow fiber membrane. Our simple conditions were conducted at 35 degrees C with no stirring and no salt addition in order to minimize sample preparation steps. Parameters such as types of hollow fiber membranes, extracting solvents and extraction time were studied and optimized. The method exhibited enrichment factors ranged from 28- to 62-fold within 30 min extraction time. The linearity of the method ranged from 0.2 to 100 microg l(-1). The limits of detection were in the low microg l(-1) level, ranging between 0.01 and 0.2 microg l(-1). The recoveries of spiked THMs at 5 microg l(-1) in water were between 98 and 105% with relative standard deviations (RSDs) less than 4%. Furthermore, the method was applied for determination of THMs in drinking water and tap water samples was reported.

Chromatography, Gas↗
PubMed2002

Streptomycetes cultured on glass beads: Sample preparation for SEM.

We demonstrate the preparation of samples of streptomycetes (Streptomyces coelicolor, S. aureofaciens) cultured on glass beads (balotina) for scanning electron microscopy. The main trick of the method consists in immobilization of glass beads with low-melting agarose. The samples are then fixed in OsO(4) vapors followed by dehydration in vapors of absolute ethanol. No air-to-liquid transition during the sample preparation occurs. Consequently, whole cell cycle of streptomycetes in the term of mycelial morphology can readily be studied by this method.

Bacteriological Techniques↗
PubMed2004

[Sample preparation methods for biomonitoring measurements of fluoride and metal content in tissues of mollusks].

Due to the toxicity of fluoride and some metals, their presence in the environment should be monitored regularly and with a simple method. The aim of this work was to analyze several methods for the preparation of tissues from mollusks prior to biomonitoring measurements of fluoride and metals. Preparation of samples for analysis requires the greatest effort and application of different procedures depending on the sample type. We present sample preparation procedures for the analysis of fluoride content in mollusk shell using the ion-selective electrode and in soft tissues using gas chromatography, as well as of metals in various mollusk tissues with atomic absorption spectrometry.

Animals↗
PubMed2006

Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins.

The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1-4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.

Carbon Isotopes↗
PubMed1999

Improved PCR detection of Campylobacter jejuni from chicken rinses by a simple sample preparation procedure.

Many food samples and enrichment media are inhibitory to the PCR, thereby lowering its detection capacity. A simple sample preparation method based on buoyant density centrifugation was examined for its application in PCR detection of Campylobacter jejuni from chicken rinse samples. Bacterial cells were spiked at different levels in a mixture of Preston broth and chicken rinse (4:1 ratio) and 0.9 ml of these mixtures were layered over 0.6 ml of gradient medium made from Percoll. PCR sensitivity for bacterial samples treated with this procedure was approximately 10-100 times higher than for samples without treatment. This sample preparation method allowed for the detection of C. jejuni from 26 of 31 naturally contaminated chicken samples after a 20-24-h enrichment period in Preston broth, compared with only 14 positives for untreated samples. In addition, the effect of Oxyrase on the growth and PCR detection of C. jejuni was examined. While Oxyrase significantly enhanced the growth and the PCR signals of C. jejuni in pure culture, it appeared not to improve the PCR detection of C. jejuni in naturally contaminated chickens.

Animals↗
PubMed2024

ZASP: A Highly Compatible and Sensitive ZnCl2 Precipitation-Assisted Sample Preparation Method for Proteomic Analysis.

Universal sample preparation for proteomic analysis that enables unbiased protein manipulation, flexible reagent use, and low protein loss is required to ensure the highest sensitivity of downstream liquid chromatography-mass spectrometry (LC-MS) analysis. To address these needs, we developed a ZnCl2 precipitation-assisted sample preparation method (ZASP) that depletes harsh detergents and impurities in protein solutions prior to trypsin digestion via 10 min of ZnCl2 and methanol-induced protein precipitation at room temperature (RT). ZASP can remove trypsin digestion and LC-MS incompatible detergents such as SDS, Triton X-100, and urea at high concentrations in solution and unbiasedly recover proteins independent of the amount of protein input. We demonstrated the sensitivity and reproducibility of ZASP in an analysis of samples with 1 μg to 1000 μg of proteins. Compared to commonly used sample preparation methods such as SDC-based in-solution digestion, acetone precipitation, FASP, and SP3, ZASP has proven to be an efficient approach. Here, we present ZASP, a practical, robust, and cost-effective proteomic sample preparation method that can be applied to profile different types of samples.

Proteomics↗
PubMed2006

Two-dimensional gel electrophoresis of peripheral nerve proteins: Optimized sample preparation.

For proteomic analysis, sample preparation plays a crucial role in two-dimensional gel electrophoresis (2DE), since, very often, each tissue or cell culture requires specific treatments. In the present paper, we report a sample preparation procedure suitable for 2DE that was done on peripheral nerve using bovine sciatic nerves and human sural nerve biopsies. We obtained an appreciable reduction of tissue heterogeneity using protein extracts obtained from nerve-fiber bundles instead of the entire nerve. In addition, we optimized 2DE protein separation using a combination of CHAPS, Triton X-100, and SB3-10 detergents in an isoelectric-focusing (IEF) buffer. The reported experimental procedures appear to be essential for 2DE separation of peripheral nerve proteins for the establishment of a reference map.

Animals↗
PubMedDate not supplied

Homogeneous sample preparation of raw shrimp using dry ice.

Sample homogeneity is critical to accurate and reproducible analysis of trace residues in foods. A method of uniform sample preparation using dry ice is described for shrimp. Other sample preparation techniques for raw shrimp produce nonhomogeneous samples. Sample homogeneity was determined through analysis of chloramphenicol added to intact tiger or white shrimp prior to sample preparation. Simulated chloramphenicol residue levels were 50, 15, 10, and 5 ppb. No significant differences were noted when analyses of shrimp inoculated with chlor-amphenicol prior to sample preparation with dry ice were compared with analyses of shrimp spiked after grinding with dry ice. Grinding shrimp with dry ice produced samples with homogeneous chloramphenicol residues. This technique should be applicable to other tissues and vegetable products.

Animals↗
PubMed2004

Solid-phase microextraction: a powerful sample preparation tool prior to mass spectrometric analysis.

Sample preparation is an essential step in analysis, greatly influencing the reliability and accuracy of resulted the time and cost of analysis. Solid-Phase Microextraction (SPME) is a very simple and efficient, solventless sample preparation method, invented by Pawliszyn in 1989. SPME has been widely used in different fields of analytical chemistry since its first applications to environmental and food analysis and is ideally suited for coupling with mass spectrometry (MS). All steps of the conventional liquid-liquid extraction (LLE) such as extraction, concentration, (derivatization) and transfer to the chromatograph are integrated into one step and one device, considerably simplifying the sample preparation procedure. It uses a fused-silica fibre that is coated on the outside with an appropriate stationary phase. The analytes in the sample are directly extracted to the fibre coating. The SPME technique can be routinely used in combination with gas chromatography, high-performance liquid chromatography and capillary electrophoresis and places no restriction on MS. SPME reduces the time necessary for sample preparation, decreases purchase and disposal costs of solvents and can improve detection limits. The SPME technique is ideally suited for MS applications, combining a simple and efficient sample preparation with versatile and sensitive detection. This review summarizes analytical characteristics and variants of the SPME technique and its applications in combination with MS.

Animals↗
PubMed2006

Miniaturized solid-phase extraction and sample preparation for MALDI MS using a microfabricated integrated selective enrichment target.

A microfabricated proteomic sample preparation and sample presentation device, Integrated Selective Enrichment Target, (ISET), comprising an array of 96 perforated nanovials is described. Each perforated nanovial can be filled with solid-phase extraction media for purification and concentration of peptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The validity of the ISET sample preparation is shown by analysis of low nM-pM standard samples, as well as biological samples. The ISET solid-phase extraction sample preparation was compared to ZipTip and MassPREP PROtarget sample preparation, demonstrating a superior performance with respect to number of detected peptides and signal intensity of detected peptides.

Caseins↗
PubMedDate not supplied

[The use of a method of solid-phase extraction for sample preparation in the identification of unknown substances by IR spectroscopy].

Identification of synthetic narcotics is difficult because they do not possess any apparent individual signs and the specimens are contaminated with various fillers and additives, which rules out investigations by infrared spectroscopy, widely used in expert practice. Use of solid-phase extraction during preparation of samples helps prepare highly purified samples which can be further identified by IR spectroscopy. The authors present the protocol of identification of unknown substances and recognition of narcotics and describe methods for identification of some confiscated narcotics (methadone, MDA, phencyclidine, methaqualone, codeine, ethylmorphine, cocaine, etc.).

Forensic Medicine↗