Biochemical Analyses of Proteins from <Emphasis Type="Italic">Duttaphrynus melanostictus</Emphasis> (<Emphasis Type="Italic">Bufo melanostictus</Emphasis>) Skin Secretion: Soluble Protein Retrieval from a Viscous Matrix by Ion-Exchange Batch Sample Preparation

A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.     

Protocol of single cells preparation for time of flight secondary ion mass spectrometry

There are several techniques like time of flight secondary ion mass spectrometry (ToF SIMS) that require a special protocol for preparation of biological samples, in particular, those containing single cells due to high vacuum conditions that must be kept during the experiment. Frequently, preparation methodology involves liquid nitrogen freezing what is not always convenient. In our studies, we propose and validate a protocol for preparation of single cells. It consists of four steps: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying under ambient conditions. The protocol was applied to samples with single melanoma cells i.e. WM115 and WM266-4 characterized by similar morphology. The surface and internal structures of cells were monitored using atomic force, scanning electron and fluorescent microscopes, used to follow any potential protocol-induced alterations. To validate the proposed methodology for sample preparation, ToF SIMS experiments were carried out using C₆₀⁺ cluster ion beam. The applied principal component analysis (PCA) revealed that chemical changes on cell surface of melanoma cells were large enough to differentiate between primary and secondary tumor sites.     

Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing

The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3-5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5-6 d for array-based enrichment and 3-4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.     

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.     

Trace-element analysis by charged-particle-induced reactions as a tool for physicians and chemists

The object of this article is to apprise physicians and chemists of nuclear analytical techniques and, in particular, of ion beam analysis (PIXE and PIGE) for the purpose of application to the clinical diagnostic method. The feasibility of the technique, sampling, and sample preparation for trace element analysis in biological and biomedical samples has been described previously (1–3). Analysis data from normal human blood samples and biomedical samples by ion beam reactions have been compared at the end. Emphasis will be placed on the use of the analytical technique on determination of the range of trace and toxic elements in human blood samples.     

Nanostructure-initiator mass spectrometry: a protocol for preparing and applying NIMS surfaces for high-sensitivity mass analysis

Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a nanostructured surface to trap liquid ('initiator') compounds. Analyte materials adsorbed onto this 'clathrate' surface are subsequently released by laser irradiation for mass analysis. In this protocol, we describe the preparation of NIMS surfaces capable of producing low background and high-sensitivity mass spectrometric measurement using the initiator compound BisF17. Examples of analytes that adsorb to this surface are small molecules, drugs, lipids, carbohydrates and peptides. Typically, NIMS is used to analyze samples ranging from simple analytical standards and proteolytic digests to more complex samples such as tissues, cells and biofluids. Critical experimental considerations of NIMS are described. Specifically, NIMS sensitivity is examined as a function of pre-etch cleaning treatment, etching current density, etching time, initiator composition, sample concentration, sample deposition method and laser fluence. Typically, NIMS surface preparation can be completed in less than 2 h. Subsequent sample preparation requires 1-5 min, depending on sample deposition method. Mass spectrometric data acquisition typically takes 1-30 s per sample.     

Determination of the efficacy of two building decontamination strategies by surface sampling with culture and quantitative PCR analysis

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus ("Bacillus subtilis subsp. niger," also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 10(5) to 10(6) CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.     

Rapid phenotypic analysis of uncoated Drosophila samples with low-vacuum scanning electron microscopy

Research projects featuring repetitive phenotypic analysis of insects, such as taxonomic studies, quantitative genetics, and mutant screens, could be greatly facilitated by a simpler approach to scanning electron microscopy (SEM). Here, we have applied low-vacuum SEM to wild type and mutant Drosophila and demonstrate that high quality ultrastructure data can be obtained quickly using minimal preparation. Adult flies, frozen live for storage, were mounted on aluminum stubs with carbon cement and directly imaged, with no chemical treatment or sputter coating. The key imaging parameters were identified and optimized, including chamber pressure, beam size, accelerating voltage, working distance and beam exposure. Different optimal conditions were found for eyes, wings, and bristles; in particular, surface features of bristles were obscured at higher accelerating voltages. The chief difficulties were charging, beam damage, and sample movement. We conclude that our optimized protocol is well suited to large-scale ultrastructural phenotypic analysis in insects.     

Analysis of polyamines in biological samples by HPLC involving pre-column derivatization with o-phthalaldehyde and N-acetyl-l-cysteine

Polyamines (putrescine, spermine and spermidine) play a crucial role in the regulation of cell growth, differentiation, death and function. Accurate measurement of these substances is essential for studying their metabolism in cells. This protocol describes detailed procedures for sample preparation and HPLC analysis of polyamines and related molecules (e.g., agmatine and cadaverine) in biological samples. The method is optimized for the deproteinization of samples, including biological fluids (e.g., 10 μl), plant and animal tissues (e.g., 50 mg), and isolated/cultured cells (e.g., 1 × 10⁶ cells). The in-line reaction of polyamines with o-phthalaldehyde and N-acetyl-l-cysteine yields fluorescent derivatives which are separated on a reversed-phase C₁₈ column and detected by a fluorometer at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total running time for each sample (including column regeneration on the automated system) is 30 min. The detection limit is 0.5 nmol/ml or 0.1 nmol/mg tissue in biological samples. The assays are linear between 1 and 50 μM for each of the polyamines. The accuracy (the nearness of an experimental value to the true value) and precision (agreement between replicate measurement) of the HPLC method are 2.5–4.2 % and 0.5–1.4 %, respectively, for biological samples, depending on polyamine concentrations and sample type. Our HPLC method is highly sensitive, specific, accurate, easily automated, and capable for the analysis of samples with different characteristics and small volume/amount, and provides a useful research tool for studying the biochemistry, physiology, and pharmacology of polyamines and related substances.     

Test of genetical isochronism for longitudinal samples of DNA sequences

Longitudinal samples of DNA sequences, the DNA sequences sampled from the same population at different time points, have increasingly been used to study the evolutionary process of fast-evolving organisms, e.g., RNA virus, in recent years. We propose in this article several methods for testing genetical isochronism or detecting significant genetical heterochronism in this type of sample. These methods can be used to determine the necessary sample size and sampling interval in experimental design or to combine genetically isochronic samples for better data analysis. We investigate the properties of these test statistics, including their powers of detecting heterochronism, assuming different evolutionary processes using simulation. The possible choices and usages of these test statistics are discussed.     

电喷雾萃取电离技术在蛋白质分析中的应用及展望

电喷雾萃取电离技术(extractive electrospray ionization,EESI)是一种能灵敏地电离固体、液体、气体、黏性样品等复杂基体中痕量大分子和小分子的新兴软电离技术．在简要介绍EESI原理的基础上,着重综述其在蛋白质分析中的应用．与商品化质谱仪器配置的电喷雾电离源(electrospray ionization,ESI)不同,EESI能够在常压条件下最大程度地保留蛋白质在样品中的原始构象,并获得大量具有生物活性的蛋白质离子．由此可见,EESI及类似的技术在蛋白质芯片制备、高分辨率氢/氘交换质谱(蛋白质结构分析)、蛋白质计量等方面具有良好的应用前景．本文也对其发展趋势进行了展望．     

Genomic DNA microextraction: a method to screen numerous samples.

Many experimental designs require the analysis of genomic DNA from a large number of samples. Although the polymerase chain reaction (PCR) can be used, the Southern blot is preferred for many assays because of its inherent reliability. The rapid acceptance of PCR, despite a significant rate of false positive/negative results, is partly due to the disadvantages of the sample preparation process for Southern blot analysis. We have devised a rapid protocol to extract high-molecular-weight genomic DNA from a large number of samples. It involves the use of a single 96-well tissue culture dish to carry out all the steps of the sample preparation. This, coupled with the use of a multichannel pipette, facilitates the simultaneous analysis of multiple samples. The procedure may be automated since no centrifugation, mixing, or transferring of the samples is necessary. The method has been used to screen embryonic stem cell clones for the presence of targeted mutations at the Hox-2.6 locus and to obtain data from human blood.     

Matrix solid phase dispersion (MSPD)

A review of the many uses of matrix solid phase dispersion (MSPD) in the extraction and analysis of a variety of compounds from a range of samples is provided. Matrix solid phase dispersion (MSPD) has found particular application as a somewhat generic analytical process for the preparation, extraction and fractionation of solid, semi-solid and/or highly viscous biological samples. Its simplicity and flexibility contribute to it being chosen over more classical methods for these purposes. MSPD is based on several simple principles of chemistry and physics, involving forces applied to the sample by mechanical blending to produce complete sample disruption and the interactions of the sample matrix with a solid support bonded-phase (SPE) or the surface chemistry of other solid support materials. These principles are discussed as are the factors to be considered in conducting a MSPD extraction.     

Opportunistic research and sampling combined with fisheries and wildlife management actions or crisis response

Currently most of the activities of state, federal, first nation, and private conservation agencies, including management of and field research on free-ranging wildlife, are not regulated under the Animal Welfare Act (AWA) and thus not subject to National Institutes of Health guidelines or routine institutional animal care and use committee (IACUC) review. However, every day thousands of fish and wildlife management activities occur across North America that provide an opportunity to take observations, measurements, biological specimens, or samples that may have research value. Most of these opportunities are secondary to ongoing and often mandated wildlife management or conservation actions. Strange as it may seem to the academic and research community, the full research potentials of these opportunities are rarely utilized. IACUCs and research institutions should strive to facilitate such research, which by its very nature is often more opportunistic than designed. They can do this by ensuring that their policies do not unnecessarily impede the rapid research responses needed, or over burden researchers with inappropriate reporting requirements designed for laboratory research. The most prominent reasons for failures to utilize wildlife research opportunities include lack of the following: personnel and expertise to collect and use the information; preparation for inevitable (or predictable) events (e.g., oil spills); resources to preserve and curate specimens; a mandate to conduct research; and recognition of the value in data or sample collection. IACUC support of open protocols and generic sampling plans can go a long way toward improving the development of useful knowledge from animals that will otherwise be lost. Opportunities to sample wildlife are categorized generally as dead sampling (road kill surveys, harvest sampling, lethal collection, and "die-offs"); live sampling (handling for marking, relocation or restocking; and captures for field or biological studies); and crisis response (e.g., population salvage operations or oil spills). Examples of the many unique situations in each category serve to illustrate how valuable research and sampling can be accomplished opportunistically. Several unique limitations of sample collection situation are described. It is recommended that IACUCs have mechanisms in place to facilitate good research in all of these circumstances.     

Optimization for peptide sample preparation for urine peptidomics

Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.     

Sample preparation issues for tissue imaging by imaging MS

Imaging MS is a powerful technique that combines the chemical and spatial analysis of surfaces. It allows spatial localization of multiple different compounds that are recorded in parallel without the need of a label. It is currently one of the rapidly developing techniques in the proteomics toolbox. Different complementary imaging MS methods, i.e. MALDI and secondary ion MS imaging for direct tissue analysis, can be applied on exactly the same tissue sample. This allows the identification of small molecules, peptides and proteins present on the same sample surface. Sample preparation is crucial to obtain high quality, reliable and reproducible complementary molecular images. It is essential to optimize the conditions for each step in the sample preparation protocol, ranging from sample collection and storage to surface modification. In this article, we review and discuss the importance of correct sample treatment in case of MALDI and secondary ion MS imaging experiments and describe the experimental requirements for optimal sample preparation.     

Matrix solid phase dispersion (MSPD)

A review of the many uses of matrix solid phase dispersion (MSPD) in the extraction and analysis of a variety of compounds from a range of samples is provided. Matrix solid phase dispersion (MSPD) has found particular application as a somewhat generic analytical process for the preparation, extraction and fractionation of solid, semi-solid and/or highly viscous biological samples. Its simplicity and flexibility contribute to it being chosen over more classical methods for these purposes. MSPD is based on several simple principles of chemistry and physics, involving forces applied to the sample by mechanical blending to produce complete sample disruption and the interactions of the sample matrix with a solid support bonded-phase (SPE) or the surface chemistry of other solid support materials. These principles are discussed as are the factors to be considered in conducting a MSPD extraction.     

A device for high-throughput monitoring of degradation in soft tissue samples

This work describes the design and validation of a novel device, the High-Throughput Degradation Monitoring Device (HDD), for monitoring the degradation of 24 soft tissue samples over incubation periods of several days inside a cell culture incubator. The device quantifies sample degradation by monitoring its deformation induced by a static gravity load. Initial instrument design and experimental protocol development focused on quantifying cartilage degeneration. Characterization of measurement errors, caused mainly by thermal transients and by translating the instrument sensor, demonstrated that HDD can quantify sample degradation with <6 μm precision and <10 μm temperature-induced errors. HDD capabilities were evaluated in a pilot study that monitored the degradation of fresh ex vivo human cartilage samples by collagenase solutions over three days. HDD could robustly resolve the effects of collagenase concentration as small as 0.5 mg/ml. Careful sample preparation resulted in measurements that did not suffer from donor-to-donor variation (coefficient of variance <70%). Due to its unique combination of sample throughput, measurement precision, temporal sampling and experimental versality, HDD provides a novel biomechanics-based experimental platform for quantifying the effects of proteins (cytokines, growth factors, enzymes, antibodies) or small molecules on the degradation of soft tissues or tissue engineering constructs. Thereby, HDD can complement established tools and in vitro models in important applications including drug screening and biomaterial development.