Standard Sample Preparation Research Articles

Adapting mass spectrometry-based platforms for clinical proteomics applications: The capillary electrophoresis coupled mass spectrometry paradigm

Single biomarker detection is common in clinical laboratories due to the currently available method spectrum. For various diseases, however, no specific single biomarker could be identified. A strategy to overcome this diagnostic void is to shift from single analyte detection to multiplexed biomarker profiling. Mass spectrometric methods were employed for biomarker discovery in body fluids. The enormous complexity of biofluidic proteome compartments implies upstream fractionation. For this reason, mass spectrometry (MS) was coupled to two-dimensional gel electrophoresis, liquid chromatography, surface-enhanced laser desorption/ionization, or capillary electrophoresis (CE). Differences in performance and operating characteristics make them differentially suited for routine laboratory applications. Progress in the field of clinical proteomics relies not only on the use of an adequate technological platform, but also on a fast and efficient proteomic workflow including standardized sample preparation, proteomic data processing, statistical validation of biomarker selection, and sample classification. Based on CE-MS analysis, we describe how proteomic technology can be implemented in a clinical laboratory environment. In the last part of this review, we give an overview of CE-MS-based clinical studies and present information on identity and biological significance of the identified peptide biomarkers providing evidence of disease-induced changes in proteolytic processing and posttranslational modification.

Using mass spectrometry to identify ubiquitin and ubiquitin‐like protein conjugation sites

Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) are polypeptides that are covalently conjugated to proteins and other biomolecules to modulate their turnover rate, localization, and/or function. The full range of Ubl functions is only beginning to be understood, and the wide variety of Ubl conjugates is only beginning to be identified. Moreover, how Ubl conjugation is regulated, and how Ubl conjugate populations change, e.g., throughout the cell cycle, in response to hormones, nutrients, or stress, or in various disease states, remains largely enigmatic. MS represents a powerful tool for the characterization of PTMs. However, standard sample preparation and data search methods are not amenable to the identification of many types of Ubl conjugates. Here, we describe the challenges of identifying Ub/Ubl conjugates, and propose an improved workflow for identification of Ub/Ubl conjugation sites. Considering the importance of Ubls in normal cellular physiology, and their roles in disease etiology and progression, it will be critical to develop improved high-throughput MS methods capable of efficiently identifying proteins and other biomolecules modified by these very interesting and important PTMs.

FemtoMolar measurements using accelerator mass spectrometry

Accelerator mass spectrometry (AMS) is an ultra-sensitive analytical method suitable for the detection of sub-nM concentrations of labeled biological substances such as pharmaceutical drugs in body fluids. A limiting factor in extending the concentration measurements to the sub-pM range is the natural (14)C content in living tissues. This was circumvented by separating the labeled drug from the tissue matrix, using standard high-performance liquid chromatography (HPLC) procedures. As the separated total drug amount is in the few fg range, it is not possible to use a standard AMS sample preparation method, where mg sizes are required. We have utilized a sensitive carbon carrier method where a (14)C-deficient compound is added to the HPLC fractions and the composite sample is prepared and analyzed by AMS. Using 50 microL human blood plasma aliquots, we have demonstrated concentration measurements below 20 fM, containing sub-amol amounts of the labeled drug. The method has the immediate potential of operating in the sub-fM region.

Rapid identification of viruses causing sugarcane mosaic by direct sequencing of RT-PCR products from crude extracts: A method for large scale virus surveys

Sugarcane mosaic virus (SCMV) and sorghum mosaic virus (SrMV) diversity studies are important to characterize virus populations in sugarcane producing areas, enabling (i) identification of shifts in predominant strains, (ii) detecting associations of strains with specific varieties, and (iii) possibly exposing the appearance of new strains which may affect the performance of varieties in a region. Recent studies have shown significant sequence variability within SCMV populations around the world, indicating that isolate identification would be best achieved by direct analysis of sequence data. Because virus sequence-based studies that require the characterization of large numbers of isolates may be impractical using standard sample preparation and processing methodology, a simple protocol that yields quality sequence information, requiring neither viral RNA purification nor cloning of RT-PCR products was developed. Rapid virus release extracts are obtained by submerging a portion of leaf tissue into an extraction buffer, followed by a brief incubation at 95 °C. An aliquot of the extract is pipetted into an RT-PCR amplification mix for the detection of SCMV and the SrMV coat protein gene fragments. RT-PCR fragments are sequenced directly using oligonucleotide primers similar to the RT-PCR primers, yielding sequence information of an adequate quality. This rapid, cost effective protocol is practical for large scale virus diversity and evolutionary studies.

Contamination of shallow groundwater system and soil–plant transfer of trace metals under amended irrigated fields

This study focuses on experimental pilot assessment of contamination of shallow groundwater systems and soil–plant transfer of trace metals under amended irrigated fields. The study approach involved a pilot experimental (greenhouse) set-up of organo-mineral amended test plots/troughs (40 cm × 47 cm × 46 cm) planted with two common vegetable crops ( Amaranthus hybridus and Abelmoschus esculentus) and irrigated with wastewater. In addition to the geochemical analyses of the primary un-amended and amended soils before planting as well as residual soils after harvesting, measurements of the physico-chemical parameters and chemical analyses of trace metals concentrations in irrigation leachates and harvested vegetable tissues were also undertaken following appropriate standard sample preparation and analytical methods. The results of the geochemical analyses carried out on irrigation leachate samples collected during the sprouting stage revealed that most of the analyzed trace metals in the collected leachates exhibited 2–10 folds depletion (except for Cu and Co with enrichment of about 1.5–3 folds) compared to the initial wastewater used for irrigation. A situation attributed to uptake/bioaccumulation of these metals and selective enrichment in the residual soils as well as to leaching by infiltrating irrigation water. Nonetheless, the observed higher trace elements concentrations in the second sets of leachates collected during harvesting stage compared to the first sets of leachates collected during the sprouting/vegetative stage is an indication of higher plant uptake during sprouting/vegetative stage or initial sorption/complexation of biosolids amendment before later vertical re-mobilization by infiltrating irrigation water. Although, virtually all of the analyzed metals exhibited elevated concentrations (2–173 ppm) in both A. hybridus and A. esculentus, a closer evaluation revealed 1.2–8.2 folds enrichment of Cr, Co, Ni, Cd, Cu, and Pb in A. esculentus compared to that of A. hybridus, an indication of the fact that phyto-accumulation of trace metal is plant-specific and dependent on physiological set-up. The overall evaluation had clearly demonstrated the potential danger of bioaccumulation of toxic trace metals under biosolid amended soils as well as impacts of irrigation-induced leaching on the shallow groundwater quality, while the need to evolve a sustainable agricultural practices is also highlighted. Capsule Organo-mineral amendment can lead to trace metal bioaccumulation (in plants) and irrigation-induced leaching to shallow groundwater system.

Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase. Apart from the catalytic Stt3p, the roles of the subunits are ill defined. Here we describe functional investigations of the Ost3/6p components of the yeast enzyme. We developed novel analytical tools to quantify glycosylation site occupancy by enriching glycoproteins bound to the yeast polysaccharide cell wall, tagging glycosylated asparagines using endoglycosidase H glycan release, and detecting peptides and glycopeptides with LC-ESI-MS/MS. We found that the paralogues Ost3p and Ost6p were required for efficient glycosylation of distinct defined glycosylation sites. Our results describe a novel method for relative quantification of glycosylation occupancy in the genetically tractable yeast system and show that eukaryotic oligosaccharyltransferase isoforms have different activities toward protein substrates at the level of individual glycosylation sites.

In Vivo Measurement of Internal and Global Macromolecular Motions in Escherichia coli

We present direct quasielastic neutron scattering measurements, in vivo, of macromolecular dynamics in Escherichia coli. The experiments were performed on a wide range of timescales to cover the large panel of internal and self-diffusion motions. Three major internal processes were extracted at physiological temperature: a fast picosecond process that corresponded to restricted jump diffusion motions and two slower processes that resulted from reorientational motions occurring in ∼40 ps and 90 ps, respectively. The analysis of the fast process revealed that the cellular environment leads to an appreciable increase in internal molecular flexibility and diffusive motion rates compared with those evaluated in fully hydrated powders. The result showed that the amount of cell water plays a decisive role in internal molecular dynamics. Macromolecular interactions and confinement, however, attenuate slightly the lubricating effect of water, as revealed by the decrease of the in vivo parameters compared with those measured in solution. The study demonstrated that standard sample preparations do not mimic accurately the physiological environment and suggested that intracellular complexity participates in functional dynamics necessary for biological activity. Furthermore, the method allowed the extraction of the self-diffusion of E. coli macromolecules, which presented similar parameters as those extracted for hemoglobin in red blood cells.

Application of PLE for the determination of essential oil components from Thymus vulgaris L.

Essential plants, due to their long presence in human history, their status in culinary arts, their use in medicine and perfume manufacture, belong to frequently examined stock materials in scientific and industrial laboratories. Because of a large number of freshly cut, dried or frozen plant samples requiring the determination of essential oil amount and composition, a fast, safe, simple, efficient and highly automatic sample preparation method is needed. Five sample preparation methods (steam distillation, extraction in the Soxhlet apparatus, supercritical fluid extraction, solid phase microextraction and pressurized liquid extraction) used for the isolation of aroma-active components from Thymus vulgaris L. are compared in the paper. The methods are mainly discussed with regard to the recovery of components which typically exist in essential oil isolated by steam distillation. According to the obtained data, PLE is the most efficient sample preparation method in determining the essential oil from the thyme herb. Although co-extraction of non-volatile ingredients is the main drawback of this method, it is characterized by the highest yield of essential oil components and the shortest extraction time required. Moreover, the relative peak amounts of essential components revealed by PLE are comparable with those obtained by steam distillation, which is recognized as standard sample preparation method for the analysis of essential oils in aromatic plants.

Qdot ® nanocrystal conjugates to expand the fluor palette for polychromatic flow cytometry

Qdot nanocrystal conjugates against leukocyte surface markers provide powerful tools to multiply fluorophore selection in flow cytometry. These semiconductor nanocrystals are best excited by UV or violet light, and provide symmetrical emission peaks 150 to 400 nm above their excitation wavelengths. Care is required with filter selection and attention to cross-laser compensation for spectral overlap. In this study, we demonstrate Qdot conjugate compatibility with standard sample preparation reagents for fixation, permeabilization, and erythrocyte lysis. We optimize filter combinations for Qdot conjugates and conventional fluors. Studies were performed on human peripheral blood leukocytes using a BD LSR II flow cytometer. Filters were optimized for nanocrystal / fluor combinations, with attention to signal intensity and compensation. For example, compensation of Qdot 605 nanocrystal emission out of the RPE-Texas Red® dye channel was reduced from ~100% to ~45% by changing from a 610 to a 620 band pass filter. As a result of individual optimizations, 8 to 10 color experiments have been run using 2 to 4 Qdot nanocrystal conjugates with good resolution of cell populations. Due to the unique spectral characteristics of nanocrystals, Qdot nanocrystal conjugates can dramatically facilitate multicolor experimental design when performing flow cytometry experiments. Supported by Invitrogen Corporation.

SCFβTrCP1 Activates and Ubiquitylates TAp63γ

p63 is a member of the p53 tumor suppressor family that is critical for epithelial differentiation and also has an important role in cancer progression. Currently, the molecular mechanisms governing regulation of p63 function remain largely unclear. This study identifies a unique E3 ubiquitin ligase for p63, SCF(betaTrCP1). SCF(betaTrCP1) is able to bind p63gamma isoforms, with a higher affinity for the TAp63gamma isoform. Strikingly, co-expression of TAp63gamma and betaTrCP1 leads to the stabilization of TAp63gamma. This stabilization of TAp63gamma leads to up-regulation of p21 at the mRNA and protein level by increased binding of TAp63gamma at the p21 promoter. The up-regulation of p21 causes a subsequent increase in G(1) phase cell cycle arrest. Last, SCF(betaTrCP1) is able to ubiquitylate TAp63gamma, and this ubiquitylation, as well as the increased activity of TAp63gamma, is ablated with the expression of a ubiquitin-deficient mutant of betaTrCP1 (DeltaFbetaTrCP1). Therefore, our study reveals that SCF(betaTrCP1) is an E3 ligase that activates p63 through ubiquitylation.

Dilution Effect in Multichannel Liquid-Handling System Equipped with Fixed Tips: Problems and Solutions for Bioanalytical Sample Preparation

This paper presents an in-depth investigation of the dilution effect from the liquid handlers equipped with fixed tips resulting from mixing with residues of the system liquid used to wash the inside and outside of the fixed tips. The impact of the dilution in bioanalytical sample preparation was evaluated. The liquid-transfer steps, where the dilution effect should be minimized or eliminated in bioanalytical sample preparation, were the sample dilution, and the standard (STD) and quality control (QC) sample preparation steps. The level of the dilution effect for the red dye was quantified by using the dual-dye photometric measurement. A significant dilution effect was observed for the red dye, indicating that without a proper liquid-transfer strategy to minimize it, it would be impossible to prepare serially diluted STD and QC samples because of the cumulative dilution effect. The impact on the bioanalytical results was substantial, especially when a multistep dilution scheme was used for the sample dilutions. A unique liquid-transfer strategy was designed in our lab to minimize the dilution effect for the sample dilution. The effectiveness of this strategy was demonstrated using the dual-dye photometric measurement and by generating a successful STD curve preparation of a model compound, Compound A, in dog plasma for an LC/MS/MS assay. With this strategy, high-quality bioanalytical sample preparation without the dilution effect could be achieved by using the liquid handlers equipped with fixed tips.

Individual variations of amitriptyline biotransformation examined in scalp hair samples.

The identification of amitriptyline, nortriptyline and its hydroxy-metabolites including a subsequent measurement of concentration profiles in hair samples was carried out to evaluate the administration history of amitriptyline. Analyses were carried out using liquid chromatography-tandem mass spectrometry, which permits simultaneous identification of all relevant substances in hair at low target concentrations (limit of detection better than 0.5pg/mg). Standard hair sample preparation was applied for the estimation of average substance concentration in a hair bundle, while segmentation of individual hairs was utilised to examine accurate concentration profiles. Replication of analyses demonstrated a good reproducibility of individual hair concentration profiles, which proved to coincide for all relevant compounds. Significant variations of metabolite ratios (e.g. nortriptyline to amitriptyline and E10- to Z10-hydroxynortriptyline) between individuals suggest a correlation between hair concentration and metabolic phenotype. Different concentration ratios of certain metabolites in hair are highly correlated, indicating a systematic association between demethylation and stereo-specificity of hydroxylation. The trans isomers of hydroxy-metabolites become significantly more prevalent with increasing degree of demethylation of amitriptyline or hydroxylation of amitriptyline.

Preparation and Standardization of Method for Valuable Metals Analysis in Seawaters

A direct analytical technique was developed and tested for correct estimation of recovery of Eco-elements such as Li, B, Br, in the seawater, and three processed seawaters from Hanjoo Co. Ltd. The reliability of recovery technique and the correct estimation of total recoveries become a major interest before the launch of pilot plant. High matrix solutions are frequently affected in analyte response during instrumental analysis as the concentration of major component changed drastically. To overcome such a complication during standard sample preparation and a better application in pilot plant, a modified matrix matching standard addition method was developed and the reliability was checked. The detection limit of elements in several seawaters analyzed by applying the modified matrix matching standard addition method were 0.144 – 0.258-g/dm3 for lithium, 0.0.013 – 0.18mg/dm3 for boron, and 4.23 – 17.8mg/dm3 for bromine. The instrumental analysis was carried by AAS for lithium and ICP-AES for boron and bromine.

Characterization of N‐palmitoylated human growth hormone by in situ liquid–liquid extraction and MALDI tandem mass spectrometry

Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N(epsilon) group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid-liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N-palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N-palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications.

A Web-Based Data Warehouse on Gene Expression in Human Malignant Melanoma

The identification of melanoma-specific dysregulated genes could identify new molecular markers. By applying bioinformatic tools for screening of biomedical databases, a melanoma-specific gene expression profile "data warehouse" was constructed. Utilizable data sets of global gene expression analyses were available from nine studies that applied different technology platforms. A single study used cell lines, five investigations analyzed cell lines and tissues obtained from patients, two studies used exclusively specimens obtained from patients, and one study analyzed blood cells prepared from patients. The total number of investigated patients was 116. From 815 differential-regulated genes, 772 (95%) were identified merely in a single study, 37 in at least two studies, five (RAB33A, ERBB3, ADRB2, MERTK, SNF1LK, and ITPKB) in at least three studies, and a single gene, RAB33A, in four studies. These data show that the accuracy, reproducibility, and comparability among different gene expression profile studies are low in melanoma. In conclusion, the study demonstrates the high diversity of gene expression profiles associated with melanoma, the necessity to include a sufficient number of samples regarding clinical standards, for the design of standardized sample collecting and preparation, for the development of common standards for microarray data processing, and for developing standardized bioinformatic tools.

Fine structure of gills and skins of the amphibious mudskipper, Periophthalmus chrysospilos Bleeker, 1852, and a non-amphibious goby, Favonigobius reichei (Bleeker, 1853)

Background. Periophthalmus chrysospilos and Favonigobius reichei are most abundant gobies species (Family: Gobiidae) inhabiting estuaries in the west coast of Peninsular Malaysia. Thirty-six goby species are amphibious: the rest remain in water, like other teleosts. However, information on the fine structure of gills and skins of these gobies species that may help to understand their functions in air breathing is still lacking. This study was aimed at describing fine structure of the gills and skins of these two co-existing species of gobies living in the terrestrial and aquatic environment. Materials and methods. Fifty-six specimens of amphibious (Periophthalmus chrysospilos) and 67 specimens of nonamphibious gobies (Favonigobius reichei) were collected from estuary. Gills and skins were dissected out and samples were treated following the standard electron microscopy sample preparation. Samples were affixed onto specimen stubs with silver paint and then coated with a thin layer of gold using a BIO-RAD-SC500 ion sputter. All samples were viewed using SEM Series XL 30, Philips. Results. P. chrysospilos possesses short, thick, bent and twisted gill filaments whereas the F. reichei has long, thin, and straight filaments. There were differences in shapes and numbers of the secondary lamellae attached to the filaments in P. chrysospilos and F. reichei. The skin of F. reichei also has a significant difference in architectural build-up compared to P. chrysospilos. The functional adaptation on the use of gills and skins in P. chrysospilos and F. reichei in natural environment are discussed. Conclusion. The gills of P. chrysospilos show some unique features (i.e. low density of secondary lamellae and smaller gill rakers) that can be used to explain the animal′s ability to successfully live out of water in comparison to F. reichei that are more adapted to aquatic life like other teleosts. Special architectural plan of gills and skins of both goby species may have contributed to their existence in the estuary area.

Factors influencing determination of shale classification indices and their correlation to mechanical properties

The influence of sample preparation procedure on the measured classification indices such as clay-size fraction, liquid limit, and plastic limit of shales was investigated. Shale samples covering a wide range of composition and plasticity were tested using standard and modified preparation procedures to yield quantitative data required for this investigation. The study specifies the range of shale plasticity in which the measured values of clay-size fraction and liquid limit are sensitive to hydrometer specimen size and rehydration time, respectively. The study also revealed that the degree of shale particle disaggregation has an important influence on the measured clay-size fraction and liquid limit. This influence is maximized for shales with intermediate plasticity and can be interpreted in terms of particle size and shape. Correlations were established between classification indices derived from standard and modified sample preparation procedures. It is recommended to use these correlations to adjust shale classification indices derived from standard sample preparation procedures usually utilized by commercial laboratories. Such correlations help in creating a universally followed procedure for better estimation of shale engineering properties and reliable interpretation of case histories involving shale formations.

Incubation of Dried and Sieved Soils Can Induce Calcium Phosphate Precipitation/Adsorption

Soil incubations are a common practice typically employed in assessing the effect of some treatment on the availability and solubility of phosphorus (P). However, standard sample preparation (drying and sieving) can alter soil chemical and physical properties, resulting in possible changes in P behavior upon soil incubation. Sixty surface soil samples were collected, air dried, and sieved before being incubated at field capacity for 7days. After incubation, soils were allowed to air dry and were analyzed along with nonincubated samples for pH and water‐ and Mehlich‐3‐extractable elements. Incubation increased pH and decreased water‐soluble P, calcium (Ca), and magnesium (Mg) relative to nonincubated soils. Increases in pH may have been due to increased solubility of residual calcium carbonates by drying and sieving. This increase in pH among soils with sufficient levels of P, Ca, and Mg resulted in the formation of Ca and Mg phosphates as confirmed by chemical speciation modeling.

Self-assembly of Alzheimer's Disease-related Amyloid Peptides into Highly Ordered Nanostructures

Alzheimer's disease (AD) related beta amyloid (Aβ) peptides possess high propensity towards aggregation. Their diffusion-controlled association follows a physico-chemically well-defined kinetics: the fibrillization starts from the monomeric/ dimeric state, and proceeds in the direction of oligomeric→protofibrillar→ fibrillar state producing neurotoxic aggregates. Nowadays one of the major directions of the drug design against AD is the synthesis of putative amyloid aggregation inhibitor molecules (AAI) which are able to hinder the formation of these toxic amyloid aggregates. Studies of both the Aβ aggregation and the effect of the AAIs on this process can be performed with several instrumental techniques. The size distribution of the aggregates up to the micron size range can be characterized with dynamic light scattering (DLS). On the other hand, species having a diameter above 5 nm can be visualized with transmission electron microscopy (TEM). In this work, we propose standardized sample preparation pro...

Complete Quantification of Group A and Group B Soyasaponins in Soybeans

A combination of high-pressure extraction and preparative chromatography was used to purify the group A and group B soyasaponins from soy germ for use as analytical standards and for use in biological assays. A standardized sample preparation and extraction method was developed for the analysis of phytochemicals found in soy and processed soy products, which is reproducible in other laboratories. The extracts can be analyzed with standard liquid chromatography-mass spectrometry and high-performance liquid chromatography methods to identify and quantitate the group A and group B forms of the soy saponins, as well as the soy isoflavones. Complete saponin analysis of the extracts prepared from soy germ (hypocots), hulls, and cotyledons shows that a significant portion of the saponins is concentrated in the germ. The germ contains nearly all of the group A soyasaponins, while the group B soyasaponins are nearly equally distributed between the germ and the cotyledons. The hulls contain little of either isoflavones or saponins. Whole (full fat) soybeans grown on a tract in central Illinois in 2003 contain approximately 4-6% saponins on a weight basis, of which about one-fifth or less of the total saponin content are group A soyasaponins; the balance is group B soyasaponins.