• Mass Spectrometry Letters
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• 제4권2호
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• pp.25-29
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• 2013

Proteomics for biomarker validation needs high throughput instrumentation to analyze huge set of clinical samples for quantitative and reproducible analysis at a minimum time without manual experimental errors. Sample preparation, a vital step in proteomics plays a major role in identification and quantification of proteins from biological samples. Tryptic digestion a major check point in sample preparation for mass spectrometry based proteomics needs to be more accurate with rapid processing time. The present study focuses on establishing a high throughput automated online system for proteolytic digestion and desalting of proteins from biological samples quantitatively and qualitatively in a reproducible manner. The present study compares online protein digestion and desalting of BSA with conventional off-line (in-solution) method and validated for real time sample for reproducibility. Proteins were identified using SEQUEST data base search engine and the data were quantified using IDEALQ software. The present study shows that the online system capable of handling high throughput samples in 96 well formats carries out protein digestion and peptide desalting efficiently in a reproducible and quantitative manner. Label free quantification showed clear increase of peptide quantities with increase in concentration with much linearity compared to off line method. Hence we would like to suggest that inclusion of this online system in proteomic pipeline will be effective in quantification of proteins in comparative proteomics were the quantification is really very crucial.

• Mass Spectrometry Letters
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• 제4권3호
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• pp.47-50
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• 2013

An ultra-fast generic LC-MS/MS method was developed for high-throughput quantification of discovery pharmacokinetic (PK) samples and its reliability was verified. The method involves a simple protein precipitation for sample preparation and the analysis by ultra-fast generic LC-MS/MS with the ballistic gradient program and selected reaction monitoring (SRM) mode. Approximately 290 new chemical entities (NCEs) (over 10,000 samples) from 5 therapeutic programs were analyzed. The calibration curves showed good linearity in the concentration range of 1, 2 or 5 to 2000 ng/mL. No significant ion suppression was observed in the elution region of all the NCEs. When approximately 300 plasma samples were continuously analyzed, the peak area of internal standard was constant and reproducible. In the repeated analysis of samples, the plasma concentrations and the area under the curve (AUC) were consistent with the results from the first analysis. These results showed that the present ultra-fast generic LC-MS/MS method is reliable in terms of selectivity, sensitivity, and reproducibility and could be useful for high-throughput quantification and other bioanalysis in drug discovery.

• 분석과학
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• 제35권5호
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• pp.218-227
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• 2022

A simple and fast throughput flow injection (FI) system with a merging-zone technique was designed to determine ferrous and ferric in an aqueous solution. The method is based on the direct reaction of ferrous with a Bathophenanthroline reagent (Bphen) in acidic media. The forming red complex absorbs light at 533 nm. All conditions of the flow injection system were investigated. The analytical curve of ferrous was linear in the range of 0.07 to 4 mg/L with an r² value of 0.9968. The detection and quantification limits were 0.02 and 0.04 mg/L, respectively. The molar absorptivity and Sandell's sensitivity were 4.0577 × 10⁶ L/mol cm and 25 × 10^-5 ㎍/cm², respectively. The homemade valve was low-cost with high repeatability (n = 7) at an RSD of 1.26 % and zero dead volume. The values of the dispersion coefficient were 2.318, 2.022, and 1.636 for the concentrations of 0.2, 1, and 3 mg/L, respectively. The analysis throughput of the designed flow injection unit was 57 sample per hour.

• Genomics & Informatics
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• 제13권4호
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• pp.119-125
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• 2015

RNA is a polymeric molecule implicated in various biological processes, such as the coding, decoding, regulation, and expression of genes. Numerous studies have examined RNA features using whole transcriptome sequencing (RNA-seq) approaches. RNA-seq is a powerful technique for characterizing and quantifying the transcriptome and accelerates the development of bioinformatics software. In this review, we introduce routine RNA-seq workflow together with related software, focusing particularly on transcriptome reconstruction and expression quantification.

• Journal of Microbiology and Biotechnology
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• 제26권2호
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• pp.213-225
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• 2016

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

• BMB Reports
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• 제37권1호
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• pp.1-10
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• 2004

DNA and RNA quantifications are widely used in biological and biomedical research. In the last ten years, many technologies have been developed to enable automated and high-throughput analyses. In this review, we first give a brief overview of how DNA and RNA quantifications are carried out. Then, five technologies (microarrays, SAGE, differential display, real time PCR and real competitive PCR) are introduced, with an emphasis on how these technologies can be applied and what their limitations are. The technologies are also evaluated in terms of a few key aspects of nucleic acids quantification such as accuracy, sensitivity, specificity, cost and throughput.

• BMB Reports
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• 제35권3호
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• pp.262-266
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• 2002

A non-isotopic neutravidin-based reverse transcriptase (RT) assay adapted for high throughput screening of HIV activity is described. Using a 96-well microtitre plate, HIV particles are lysed and the RT enzyme released into a reaction mixture containing poly(A) RNA, biotinylated oligo d(T) and fluorescein-labelled dUTP (FI-dUTP). With poly(A) as a template and oligo d(T) as primer, the viron RT incorporates FI-dUTP into an elongating DNA strand. The resulting product is captured on a neutravidin-coated 96-well plate and the unincorporated nucleotides removed by a series of washing steps. A simple ELISA is subsequently performed using a monoclonal antifluorescein antibody conjugated to alkaline phosphatase. Quantification of RT activity is facilitated by a colorimetric readout. The assay was validated in the context of a diagnostic HIV-1 phenotyping assay. Using supernatants from HIV-1 infected lymphocyte cultures the assay was shown to be as sensitive as a radioactive assay and the RT activity correlated well with levels of cell-asociated HIV-p24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.

• 한국소성가공학회:학술대회논문집
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• 한국소성가공학회 2004년도 추계학술대회논문집
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• pp.305-310
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• 2004

The Hot Embossing Lithography(HEL) as a method for the fabrication of nanostructure with polymer is becoming increasingly important because of its simple process, low cost, high replication fidelity and relatively high throughput. In this study, we investigated the characteristics of hot embossing lithography as a nanoreplication technique. To grasp characteristics of nano patterning rheology by process parameters(embossing temperature, pressure and time), we have carried out various experiments by using the DVD(400nm pattern width) and Blu-ray nickel stamps(150nm pattern width). During the hot embossing process, we have observed the characteristics of the size effect. The quality of products made by hot embossing is affected by its cooling shrinkage. The demolding process at the glass transition temperature results in low quality because of the shrinkage of the polymer. Therefore, the quantification of the temperature condition is essential for the replication of high quality.

• Mass Spectrometry Letters
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• 제2권3호
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• pp.69-72
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• 2011

Defective synthesis of the steroid hormones by the adrenal cortex has profound effects on human development and homeostasis. Due to the time-consuming chromatography procedure combined with mass spectrometry, the matrix-assisted laser desorption ionization method coupled to the linear ion-trap tandem mass spectrometry (MALDI-LTQ-MS/MS) was developed for quantitative analysis of 10 adrenal steroids in human serum. Although MALDI-MS can be introduced for its applicability as a high-throughput screening method, it has a limitation on reproducibility within and between samples, which renders poor reproducibility for quantification. For quantitative MALDI-MS/MS analysis, the stable-isotope labeled internal standards were used and the conditions of crystallization were tested. The precision and accuracy were 3.1~35.5% and 83.8~138.5%, respectively, when a mixture of 10 mg/mL ${\alpha}$-cyano-4-hydroxycinnamic acid in 0.2% TFA of 70% acetonitrile was used as the MALDI matrix. The limit of quantification ranged from 5 to 340 ng/mL, and the linearity as a correlation coefficient was higher than 0.988 for all analytes in the calibration range. Clinical applications include quantitative analyses of patients with congenital adrenal hyperplasia. The devised MALDI-MS/MS technique could be successfully applied to diagnosis of clinical samples.

• Journal of Microbiology and Biotechnology
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• 제27권7호
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• pp.1345-1358
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• 2017

The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N-acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.