Absolute Protein Concentrations Research Articles

Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins.

The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omics" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.

Abstract 1334: Dual-isolation of CTCs and cancer exosomes from blood samples with melanoma using immunoaffinity based OncoBean microfluidic devices

Abstract Introduction: Melanoma is among the most aggressive cancers, and its incidence continues to grow. Due to the lack of promising markers to predict the disease and onset of metastasis, early detection and evaluation of treatment efficacy have been hampered. Recent advances in liquid biopsy have proposed alternatives for diagnosing disease with the merits of enabling continuous monitoring and non-invasiveness. CTCs and cancer exosomes are evolving as promising biomarkers due to their innate capability of having cancer-associated molecules and signatures. However, simultaneous isolation of CTCs and exosomes using the same methods from the identical samples has not been studied yet. Here, we propose the use of the OncoBean microfluidic device conjugated with melanoma specific antibodies, MCAM and MCSP for the simultaneous isolation. Using whole blood samples from patients, CTCs and exosomes are specifically isolated from the same samples and then undergo molecular profiling for comprehensive studies. Methods: OncoBean devices were fabricated by soft lithography using polydimethylsiloxane (PDMS). The fabricated devices were conjugated with biotinylated MCSP and MCAM, using Neutralavidin-biotin chemistry. Using 6ml blood samples, 3ml of whole blood and 3ml of plasma were used for CTC isolation and melanoma-associated exosome isolation, respectively. In order to remove cellular debris, the plasma sample was filtered using a 200nm filter. After CTC isolation, captured cells were stained with fluorescent antibodies for melanoma specific (Melan-A and S100), leukocyte (CD45), and nucleus (DAPI) markers. The captured and stained CTCs were enumerated with consideration of size, nucleus to cytoplasm ratio and melanoma marker expression. The isolated exosomes were examined by scanning electron microscopy (SEM) to evaluate their abundance and size. The protein concentration and exosomal protein expression were evaluated by protein extraction using RIPA buffer. Results: All melanoma patient samples showed 1-35 CTCs per 3ml of blood. Healthy donors had no CTCs. The abundance of isolated exosomes was evaluated by SEM and showed sizes ranging from 117-143nm. The average exosomal protein from melanoma was 28.6±19.2μg/ml, considerably higher than that of healthy donor, 11.0±3.1μg/ml. The exosomes isolated by OncoBean tested positively for the common exosomal protein, CD9, implying that the present device isolated melanoma exosomes specifically. Discussion and conclusion: We showed that melanoma patients have both circulating tumor cells and cancerous exosomes in their blood samples. Even though their absolute CTC number and exosomal protein concentration showed no high correlation, simultaneous isolation using the identical microfluidic devices will be helpful for further verification of disease and cross validation according to their roles in disease. Citation Format: Yoon-Tae Kang, Ting-Wen Lo, Thomas Hadlock, Emma Purcell, Aaron Kramer, Monica De Reguera, Scott Alan McLean, Sunitha Nagrath. Dual-isolation of CTCs and cancer exosomes from blood samples with melanoma using immunoaffinity based OncoBean microfluidic devices [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1334.

The Potential Role of Protein Leverage in the US Obesity Epidemic.

The protein leverage model of obesity posits that decreasing the protein fraction of the diet leads to compensatory increases in total energy intake in an attempt to maintain a target amount of absolute protein consumed. The resulting increased energy intake thereby causes weight gain. According to food balance sheets published by the Food and Agriculture Organization of the United Nations, while the absolute protein content of the US food supply has increased since the early 1970s, the fraction of available calories from protein has decreased by ~1% because of greater increases in available carbohydrate and fat. Counterintuitively, even such a small decrease in the protein fraction of the food supply has the potential to result in relatively large increases in energy intake according to the protein leverage model. Therefore, while the protein leverage effect is unlikely to fully explain the obesity epidemic, its potential contribution should not be ignored.

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Human Stem Cell Structures Measured with Concentration-Calibrated Super-Resolution Microscopy

The mission of the Allen Institute for Cell Science is to understand and predict cell behaviors. The current project aims to create a stem cell state space by conjoining genomic, imaging, and phenotypic data from cells. We tagged most major molecular machineries (organelles, membranes and cytoskeleton) of human induced pluripotent stem cells (hiPSCs) with fluorescent proteins, and acquired large, reproducible datasets of each cell line for model input using an automated spinning-disk microscopy pipeline (www.allencell.org). We used the CRISPR/Cas9 system to fuse the fluorescent proteins directly to the protein of interest under the endogenous promoter. Therefore, the measured intensity of fluorescence emission is proportional to the protein distribution, localization, and dynamics of individual cells. To improve the accuracy of the integrated cell models we are building, we collected data with higher spatial resolution and measured absolute protein concentrations. We increased the resolution by about 1.7x using the Zeiss LSM 880 confocal microscope equipped with an Airy fast detector based on pixel reassignment. We created intensity/concentration calibration standards using cytosolic eGFP, cell-lysate, as well as purified eGFP in solution. We calibrated the concentration standards with FCS (Fluorescence Correlation Spectroscopy). The concentration standards allow quantitative comparison of data collected on different systems. We validated these approaches with quantitative Western blots, ELISA, and UV/VIS spectroscopy. We will discuss the advantages and limitations of the different standards, as well as the limitations, corrections, and controls necessary because of mono-allelic expression of eGFP-tagged proteins, inactive eGFP, and environmental changes of eGFP brightness. These results will add additional, quantitative constraints to our integrated models based on concentrations as well as finer structural detail due to the improved resolution.

Measuring Protein Concentration by Diffusion-Filtered Quantitative Nuclear Magnetic Resonance Spectroscopy.

The concentration of macromolecules in solution is a crucial property in many areas of research, including the development and commercialization of biological therapeutics. For proteins in particular, none of the reported methods for measuring concentration detect a molecular property that is known a priori; rather, they rely on ligand binding, degradation and derivitization, or an intrinsic property that must be determined experimentally. The purpose of this report is to describe (1) a diffusion-filtered qNMR experiment (DF-qNMR) for quantitating macromolecules in complex matrices and (2) an overall method for measuring absolute protein concentration based on this DF-qNMR experiment. This method combines protein denaturation with the diffusion filter to produce clean spectra of the protein with well-resolved resonances, regardless of the matrix complexity. The concentration is then obtained by comparing the peak area of the valine/isoleucine/leucine methyl groups to an external, certified, small-molecule quantitation standard. The method, which is referred to as VILMHA (valine isoleucine leucine methyl hydrogen analysis), was tested on three proteins of various sizes. In all cases, the measured concentration was within 1.8% of the labeled value for the undiluted standard reference material evaluated. In addition, the RSD's were less than 1.25% in all cases and less than 1% in most cases. The accuracy, precision, and ease of use make this method superior to existing absolute protein concentration methods. Furthermore, VILMHA is ideally suited to serve as the basis for converting the relative protein concentration methods into absolute methods or establishing molecular-specific parameters. Finally, DF-qNMR has the potential to quantitate other types of macromolecules (e.g., such as polymers, surfactants, etc.) in the presence of small-molecule contaminants.

Selection of Early Soybean Inbred Lines Using Multiple Indices

Soybean [Glycine max (L.) Merr.] has been studied and enhanced for most of its economically important traits. Previous research has studied the association among them and the effect of the genotype × environment interaction; however, less is known about their correlation considering absolute maturity, as well as the use of multiple selection indices to study the genotype × environment interaction and select superior cultivars. Regarding this, the aim of the present study was to identify lines that associate precocity, good yield performance, and high oil and protein contents in the grains, as well as to estimate the correlation among these traits and study the effect of genotype × environment interaction using a standardized multiple selection index. Trials were conducted in two crop seasons in the state of Minas Gerais, Brazil, with 39 lines in 13 evaluation environments. The experiments were conducted in a randomized complete block design with four replications, and the grain yield, absolute maturity, and oil and protein contents in the grains were evaluated. The results indicated high experimental precision and accuracy, with significant differences among lines for all traits. High‐magnitude correlations between evaluated traits were found, highlighting the negative correlation between absolute maturity and protein content in the grains. The genotype × environment interaction was also significant, and the use of the multiple selection index was efficient to identify superior and stable inbred lines by the genotype + genotype × environment (GGE) biplot method, which explained 82.23% of the genotype × environment interaction effect. Lines 27 and 31 stood out from the others because they associated stability and good performance for all evaluated traits.

Content and Activities of UGT2B7 in Human Liver In Vitro and Predicted In Vivo: A Bottom-Up Approach

UDP-glucuronosyltransferase 2B7 (UGT2B7) is one of the most significant isoforms of UGTs in human liver. This research measured UGT2B7 protein content and activities, including maximum velocity (Vmax) and intrinsic clearance (CLint), in human liver at isoform, microsomal, liver tissue, and liver levels and identified the factors that influence expression. We determined absolute protein content by liquid chromatography-tandem mass spectroscopy and activities using the probe drug zidovudine in 82 normal human liver microsomes. Using a bottom-up method for derivation, we showed UGT2B7 content at the microsomal, liver tissue, and liver levels, as well as activities at the isoform, microsomal, liver tissue, and liver levels in vitro, and predicted hepatic clearance in vivo, with median, range, variation, and 95% and 50% prediction intervals. With regard to the intrinsic activities, the maximum velocity (Vmax) had a median (range) of 7.5 (2-24) pmol/min per picomole of 2B7, and the CLint was 0.08 (0.02-0.31) μl/min per picomole of 2B7. Determinations at liver level showed larger variations than at microsomal level, so it was more suitable for evaluating individual differences. By analyzing factors that affect UGT2B7, we found that: 1) The content at the liver tissue and liver levels correlated positively with activities; 2) the mutant heterozygotes of -327G>A, -900A>G, -161C>T may lead to decreased protein content and increased intrinsic CLint; and 3) the transcription factor pregnane X receptor mRNA expression level was positively associated with the measured protein content. In all, we showed that protein content and activities at different levels and the factors that influence content provide valuable information for UGT2B7 research and clinically individualized medication.

Precise determination of protein extinction coefficients under native and denaturing conditions using SV-AUC.

The accurate determination of protein concentration is an important though non-trivial task during the development of a biopharmaceutical. The fundamental prerequisite for this is the availability of an accurate extinction coefficient. Common approaches for the determination of an extinction coefficient for a given protein are either based on the theoretical prediction utilizing the amino acid sequence or the photometric determination combined with a measurement of absolute protein concentration. Here, we report on an improved SV-AUC based method utilizing an analytical ultracentrifuge equipped with absorbance and Rayleigh interference optics. Global fitting of datasets helped to overcome some of the obstacles encountered with the traditional method employing synthetic boundary cells. Careful calculation of dn/dc values taking glycosylation and solvent composition into account allowed the determination of the extinction coefficients of monoclonal antibodies and an Fc-fusion protein under native as well as under denaturing conditions. An intra-assay precision of 0.9% and an accuracy of 1.8% compared to the theoretical value was achieved for monoclonal antibodies. Due to the large number of data points of a single dataset, no meaningful difference between the ProteomeLab XL-I and the new Optima AUC platform could be observed. Thus, the AUC-based approach offers a precise, convenient and versatile alternative to conventional methods like total amino acid analysis (AAA).

ИСПОЛЬЗОВАНИЕ ПОКАЗАТЕЛЕЙ ОТНОСИТЕЛЬНОГО И АБСОЛЮТНОГО СОДЕРЖАНИЯ БЕЛКА В ЗЕРНЕ ОЗИМОЙ ПШЕНИЦЫ ПРИ СЕЛЕКЦИИ НА КАЧЕСТВО

The use of the indexes of relative and absolute content of protein in winter wheat grain selected on quality Winter wheat is the main food crop, which occupies a significant proportion in the structure of the grain area of Russia. The crop uses bioclimatic potential of the regions of cultivation better than spring wheat and supplies stable grain production. Winter wheat on Don supplies up to 50% of gross grain, and sometimes up to 70%. The article presents the study results of the accumulation of relative and absolute protein content in grain depending upon 1000-kernel weight on average during the years of 2014-2016. The obtained data indicate that absolute protein content in grain does not depend on the productivity. This index is really stable. Such indexes as 1000-kernel weight, relative protein content in grain, gross yield of protein per hectare, winter wheat productivity are significantly changeable. Protein content in grain is slightly affected by weather conditions. There is an inverse relation between 1000-absolutely dry kernel weight and relative protein content in grain. The more 1000-absolutely dry kernel weight is, the less protein content in grain is and vice versa. But there are significant differences of the trait among the varieties. The varieties “Rostovchanka 5” and “Rostovchanka 7” with less 1000-kernel weight than the varieties “Tanais”, “Aksiniya” and “Nakhodka”, have formed less protein content through all the years. The varieties “Tanais”, “Aksiniya” and “Nakhodka” with 1000-kernel weight of 41.1-43.7 g produced the largest amount of protein (14.7%, 14.4% and 14.2% respectively).

Absolute Protein Quantification Using AQUA-Calibrated 2D-PAGE.

Absolute protein quantification for the analysis of proteome dynamics is more and more required by the scientific community. Therefore a number of methods have recently been reported that aim at determining concentrations of single proteins in complex samples, all of them having their advantages and limitations. However, for all of these methods an accurate and protein unspecific determination of the total protein amount in a given sample is urgently needed. Here a ninhydrin-based assay established to reach this goal is described. Moreover, an optimized protocol for protein digestion is an inevitable prerequisite for all mass spectrometry-based approaches aiming at absolute protein quantification. In this chapter, various aspects are described which have to be considered during validation of a suitable digestion method and a detailed protocol is presented that can be applied to the digestion of soluble proteins originated from microbes.In order to provide an absolute protein quantification workflow applicable for small scale and large scale approaches, a step-by-step guide is provided for the so-called AQUA-strategy (AQUA=absolute quantification), including selection of suited standard peptides, the development of optimized MS methods and the determination of absolute protein concentration using stable isotope dilution and selected reaction monitoring (SID-SRM). Subsequently, a workflow is introduced that combines targeted mass spectrometry and two-dimensional polyacrylamide gel electrophoresis for the large-scale determination of absolute protein amounts.

Algorithm of targeted (SRM) methods development in mass-spectrometric studies

Determination of protein concentration in biological samples is an important task for biological research, as well as for medicine and routine clinical biochemistry. The introduction of stable isotope-labeled peptide standards (SIS) made it possible to determine accurately absolute protein concentrations in proteomic studies. The correct choice of SIS and the systematic way to develop a s method for selected reaction monitoring (SRM) are very important steps that are crucial for further identification and measurements of protein concentration. In this paper, we summarize our experience of selecting SIS for measuring the protein concentration by SRM. The results are presented in the form of an algorithm that describes the main stages of the SIS selection and the main points in the development of SRM methods for the targeted protein detection and determination of protein concentrations in biological samples.

59 Selected Reaction Monitoring-Based Absolute Quantification of Developmentally Relevant Proteins in Early Bovine Embryos Reveals Differences Between In Vitro and In Vivo Embryo Culture and Between Different Maternal Metabolic Stages

Early embryogenesis is a highly complex developmental process, accompanied by a plethora of changes at the morphological and molecular level. Particularly at the level of proteins, these changes are still poorly characterised and understood. During the first cleavages, the embryo depends mainly on maternal transcripts and proteins that were accumulated and stored during oogenesis until embryonic genome activation (EGA) occurs. In the bovine system, the major EGA takes place at the 8- to 16-cell stage. However, we recently demonstrated by liquid chormatography-tandem mass spectrometry (LC-MS/MS)-based holistic proteome approaches that despite transcriptional and translational silencing, the proteome of the early embryo is highly dynamic (Deutsch et al. 2014; Demant et al. 2015). Based on these findings, we established a targeted LC-MS/MS approach based on multiplexed selected reaction monitoring (mSRM), which facilitates an absolute quantification of 27 proteins relevant in early embryogenesis. Each protein is targeted by 2 independent peptides to facilitate highly reliable quantifications. Nine characteristic developmental stages from germinal vesicle oocyte to hatched blastocyst were analysed (n = 6 per stage), and absolute protein contents are reported as femtomole per embryo, with limits of quantification (LOQ) down to 100 attomoles per embryo. Based on their abundance profiles during maturation, zygote formation, and embryonic development, the 27 proteins could be grouped into 6 SOTA clusters. By principal component analysis (PCA), absolute SRM quantifications of only 9 selected proteins were shown to discriminate between all 9 developmental stages analysed, thus providing molecular fingerprints significant for each developmental stage. We used the 27-plex SRM assay as a powerful readout tool and demonstrated substantial quantitative differences between embryos derived from a well-established in vitro culture system and embryos transferred into the oviduct of living animals for 2 days (in vivo culture). Furthermore, in vivo development of embryos in animals differing in their metabolic stress levels led to significant alterations in the 27-plex SRM profiles. This work was supported by a grant to GJA from Deutsche Forschungsgemeinschaft DFG FOR1041 ‘Germ Cell Potential’ AR 362/7-1 and European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 312097 - FECUND.

Proof of the quantitative potential of immunofluorescence by mass spectrometry

Protein expression in formalin-fixed, paraffin-embedded patient tissue is routinely measured by Immunohistochemistry (IHC). However, IHC has been shown to be subject to variability in sensitivity, specificity and reproducibility, and is generally, at best, considered semi-quantitative. Mass spectrometry (MS) is considered by many to be the criterion standard for protein measurement, offering high sensitivity, specificity, and objective molecular quantification. Here, we seek to show that quantitative immunofluorescence (QIF) with standardization can achieve quantitative results comparable to MS. Epidermal growth factor receptor (EGFR) was measured by quantitative immunofluorescence in 15 cell lines with a wide range of EGFR expression, using different primary antibody concentrations, including the optimal signal-to-noise concentration after quantitative titration. QIF target measurement was then compared to the absolute EGFR concentration measured by Liquid Tissue-selected reaction monitoring mass spectrometry. The best agreement between the two assays was found when the EGFR primary antibody was used at the optimal signal-to-noise concentration, revealing a strong linear regression (R2=0.88). This demonstrates that quantitative optimization of titration by calculation of signal-to-noise ratio allows QIF to be standardized to MS and can therefore be used to assess absolute protein concentration in a linear and reproducible manner.

The Levels of C-Reactive Protein, Malondialdehyde and Absolute Lymphocyte Counts in Pre and Post-Acute Exercise

This study was designed to determine the level of oxidative stress using the level of circulating C-reactive protein, malondialdehyde and absolute lymphocyte counts in pre and post-acute exercise as studies have shown that acute exercise enhances the immunological responses of stressed individuals. Twenty five (25) healthy young male undergraduate students with an average age of 24.3 ± 3 years and body mass index of 22.7 ± 1.8 (kg/m2) participated fully in the study. The levels of malondialdehyde and C-reactive protein were significantly higher at one hour, four hours and twenty-four hour post exercise when compared with the pre-exercise stage whereas the absolute lymphocyte count and absolute neutrophils count were significantly higher at one hour and four hours post exercise when compared with the pre-exercise stage. Absolute lymphocyte count, absolute neutrophils count, Creactive protein and malondialdehyde concentrations are increased in acute exercise which is an indication of an acute phase responses during stressful events.

Plasma protein absolute quantification by nano-LC Q-TOF UDMSE for clinical biomarker verification.

Background and aimsProteome-based biomarker studies are targeting proteins that could serve as diagnostic, prognosis, and prediction molecules. In the clinical routine, immunoassays are currently used for the absolute quantification of such biomarkers, with the major limitation that only one molecule can be targeted per assay. The aim of our study was to test a mass spectrometry based absolute quantification method for the verification of plasma protein sets which might serve as reliable biomarker panels for the clinical practice.MethodsSix EDTA plasma samples were analyzed after tryptic digestion using a high throughput data independent acquisition nano-LC Q-TOF UDMSE proteomics approach. Synthetic Escherichia coli standard peptides were spiked in each sample for the absolute quantification. Data analysis was performed using ProgenesisQI v2.0 software (Waters Corporation).ResultsOur method ensured absolute quantification of 242 non redundant plasma proteins in a single run analysis. The dynamic range covered was 105. 86% were represented by classical plasma proteins. The overall median coefficient of variation was 0.36, while a set of 63 proteins was found to be highly stable. Absolute protein concentrations strongly correlated with values reviewed in the literature.ConclusionsNano-LC Q-TOF UDMSE proteomic analysis can be used for a simple and rapid determination of absolute amounts of plasma proteins. A large number of plasma proteins could be analyzed, while a wide dynamic range was covered with low coefficient of variation at protein level. The method proved to be a reliable tool for the quantification of protein panel for biomarker verification in the clinical practice.

Addition of Amylase from Aspergillus Awamori to the Diet of Broiler Chickens

Two experiments were performed to evaluate the hematological and blood biochemistry parameters, biometry of digestive organs, enzyme activities, protein content and absolute weight of the pancreas of broilers fed pre-starter and pre-starter diets supplemented or not with amylase from Aspergillus awamori. In total, 120 male Cobb chicks were housed in heated cages in each experiment. A completely randomized experimental design, with two treatments (feed with and without amylase) and six replicates per treatment of 10 birds each was applied. The data were subjected to analysis of variance using the F-test at 5% probability level. The dietary amylase addition did not affect hematological and blood biochemistry parameters and the biometry of the gastrointestinal tract of 7- and 21-d-old broilers, nor the absolute weight, enzyme activities or protein concentration of the pancreas of 7-d-old broilers. However, the inclusion of amylase in the diet reduced amylase activity and pancreatic protein concentration in 21-d-old broilers. The application of amylase to broiler chicken pre-starter and starter feeds is not justified given the pancreatic amylase activity and protein concentrations.

Inference Method for Developing Mathematical Models of Cell Signaling Pathways Using Proteomic Datasets.

The progress in proteomics technologies has led to a rapid accumulation of large-scale proteomic datasets in recent years, which provides an unprecedented opportunity and valuable resources to understand how living organisms perform necessary functions at systems levels. This work presents a computational method for designing mathematical models based on proteomic datasets. Using the mitogen-activated protein (MAP) kinase pathway as the test system, we first develop a mathematical model including the cytosolic and nuclear subsystems. A key step of modeling is to apply a genetic algorithm to infer unknown model parameters. Then the robustness property of mathematical models is used as a criterion to select appropriate rate constants from the estimated candidates. Moreover, quantitative information such as the absolute protein concentrations is used to further refine the mathematical model. The successful application of this inference method to the MAP kinase pathway suggests that it is a useful and powerful approach for developing accurate mathematical models to gain important insights into the regulatory mechanisms of cell signaling pathways.

Ethanol-metabolizing activities and isozyme protein contents of alcohol and aldehyde dehydrogenases in human liver: phenotypic traits of the ADH1B*2 and ALDH2*2 variant gene alleles.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for the metabolism of ethanol. East Asian populations are unique in that they carry both a prevalent ADH1B*2 and a dominant-negative ALDH2*2 allele. A systematic investigation of ethanol-metabolizing activities in normal livers correlated with the corresponding functional allelic variations and protein contents of the relevant isozymes in respective enzyme families has been lacking. To obtain a reasonable sample size encompassing all possible genetic allelotypes of the ADH1B and ALDH2, 141 surgical liver specimens from adult Han Chinese were studied. Expression patterns and activities of ADH and ALDH were determined with stratification of the genetic phenotypes. Absolute protein contents as well as cellular localization of the activity and protein of ADH/ALDH isozymes were also investigated. The activities of ADH1B*1/*2 and ADH1B*2/*2 allelic phenotypes were 5-6-fold those of the ADH1B*1/*1, suggesting that ADH1B*2 allele-encoded subunits are dominant over expression of hepatic ADH activity. The activities of the ALDH2-active phenotype were 90% higher than those of the ALDH2-inactive phenotype. Sex and age did not significantly influence the hepatic ADH and ALDH activities with specified genetic phenotypes. The isozyme protein contents were as follows in decreasing order: ADH1, ADH2, ALDH1A1, ALDH2, and ADH3. Both ADH1, but not ADH2/3, and ALDH1A1/2 showed a preferential expression in perivenular hepatocytes. Functional correlations of ADH1B*2 and ALDH2*2 variant alleles in the liver provide a biochemical genetic basis suggesting their contribution toward variability in ethanol metabolism as well as susceptibility to alcoholism and alcohol-related diseases in East Asians.

Nutrient restriction and realimentation in beef cows during early and mid-gestation and maternal and fetal hepatic and small intestinal in vitro oxygen consumption

Objectives were to determine the effects of advancing gestation, maternal nutrient restriction during early and mid-gestation, and realimentation on fetal liver and jejunal mass and energy use in both dams and fetuses. On day 30 of pregnancy, multiparous, non-lactating beef cows (initial BW=621±11.3 kg and body condition score=5.1±0.1) were assigned to one of the two dietary treatments: control (CON; 100% requirements; n=18) and restricted (R; 60% requirements; n=28). On day 85, cows were slaughtered (CON, n=6; R, n=6), and remaining cows continued on control (CC; n=12) and restricted (RR; n=12) diets, or were realimented to the control diet (RC; n=11). On day 140, cows were slaughtered (CC, n=6; RR, n=6; RC, n=5), remaining cows continued on the control diet (CCC, n=6; RCC, n=5), or were realimented to the control diet (RRC, n=6). On day 254, all remaining cows were slaughtered. Maternal liver O2 consumption linearly increased (P⩽0.04) and jejunal weight (g/kg) linearly decreased (P=0.04) as gestation advanced in CON groups. Fetal BW, and hepatic and small intestinal absolute mass, protein content and O2 consumption linearly increased (P⩽0.04) as pregnancy advanced in CON groups. However, mass and O2 consumption relative to BW linearly decreased (P⩽0.001) in the fetal liver in CON groups. When analyzing the effects of dietary treatment, at day 85, fetal jejunal O2 consumption (mol/min per kg BW) was lower (P=0.02) in the R group when compared with the CON group. At day 140, maternal hepatic weight (g) was lower (P=0.02) in RC and RR cows when compared with CC, and fetal jejunual O2 consumption (mmol/min per mg tissue and mmol/min per g protein) was greater (P⩽0.02) in RC when compared with RR. At day 254, maternal hepatic O2 consumption (absolute and relative to BW) was lower (P⩽0.04) in the RCC cows when compared with RRC. Fetal hepatic weight was lower (P=0.05) in the CCC group when compared with RCC and RRC. The changes in response to nutrient restriction and realimentation in both the dam and fetus may indicate an adaptation to a lower amount of available nutrients by altering tissue mass and metabolism.