Milk Proteomics

Milk proteins have been studied continuously for over 50 years. Knowledge of this complex protein system has evolved incrementally in recent decades, largely coinciding with advances in technology. Proteomics and associated technologies have the potential to facilitate further advances in our knowledge of milk proteins. Proteomics allows for the detection, identification and characterization of milk proteins. More importantly, proteomics facilitates the analysis of large numbers of milk proteins simultaneously. In the first part of this review we provide a description of the key techniques used within proteomic methodologies, with an emphasis on their general uses within proteomics. In the second part we summarize recent applications of proteomics to milk proteins and highlight the potential for new and rapid advances in the analysis of milk proteins. In particular, we emphasise the effectiveness of two-dimensional gel electrophoresis in combination with various mass spectrometry techniques for the detailed characterization of milk proteins. (C) 2004 Elsevier Ltd. All rights reserved.

Identification of a novel frameshift mutation in the giant muscle filament titin in a large Australian family with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is an etiologically heterogeneous cardiac disease characterized by left ventricular dilation and systolic dysfunction. Approximately 25-30% of DCM patients show a family history of mainly autosomal dominant inheritance. We and others have previously demonstrated that mutations in the giant muscle filament titin (TTN) can cause DCM. However, the prevalence of titin mutations in familial DCM is unknown. In this paper, we report a novel heterozygous 1-bp deletion mutation (c.62890delG) in TTN that cosegregates with DCM in a large Australian pedigree (A3). The TTN deletion mutation c.62890delG causes a frameshift, thereby generating a truncated A-band titin due to a premature stop codon (p.E20963KfsX10) and the addition of ten novel amino acid residues. The clinical phenotype of DCM in kindred A3 demonstrates incomplete penetrance and variable expressivity. Finally, protein analysis of a skeletal muscle biopsy sample from an affected member did not reveal the predicted truncated titin isoform although the aberrant mRNA was present, suggesting posttranslational modification and degradation of the truncated protein. The identification of a novel disease-causing mutation in the giant titin gene in a third large family with DCM indicates that mutations in titin may account for a significant portion of the genetic etiology in familial DCM.

Neurobiological alterations in the human alcoholic brain: effect of genotype

Long-term alcohol abuse by human subjects leads to selective brain damage that is restricted in extent and variable in severity. Within the cerebral cortex, neuronal loss is most marked in the superior frontal cortex and relatively mild in motor cortex. Cirrhotic alcoholics and subjects with alcohol-related Wernicke-Korsakoff syndrome show more severe and more extensive damage than do uncomplicated cases. Accumulating evidence suggests that the likelihood of developing alcohol dependency is associated with one or more genetic markers. In previous work we showed that GABAA receptor functionality, and the subunit isoform expression that underlies this, differed in region- and disease-specific ways between alcoholics and controls. By contrast, glutamate receptor (NMDA, KA, AMPA) differences were muted or absent. Here we asked if genotype differentiated the form, pharmacology, or expression of glutamate and GABA receptors in pathologically vulnerable and spared cortical regions, with a view to determining whether such subject factors might influence the severity of alcohol-induced brain damage. Cerebrocortical tissue was obtained at autopsy under informed, written consent from uncomplicated and alcoholic-cirrhotic Caucasian (predominantly Anglo-Celtic) cases, together with matched controls and cases with cirrhosis of non-alcoholic origin. All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were processed for synaptic membrane receptor binding, mRNA analysis by quantitative RT-PCR, and protein analysis by Western blot. Genotyping was performed by PCR methods, in the main using published primers. Several genetic markers differentiated between our alcoholic and control subjects, including the GABAA receptor 2 subunit (GABB2) gene ( 2 (3) 10.329, P 0.01), the dopamine D2 receptor B1 (DRD2B) allele ( 2 (3) 10.109, P 0.01) and a subset of the alcohol dehydrogenase-3 (ADH3) alleles ( 2 (2) 4.730, P 0.05). Although neither the type-2 glutamate transporter (EAAT2) nor the serotonin transporter (5HTT) genes were significantly associated with alcoholism, only EAAT2 heterozygotes showed a significant association between ADH3 genotype and alcoholism ( 2 (3) 7.475, P 0.05). Other interactions between genotypes were also observed. DRD2A, DRD2B, GABB2, EAAT2 and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters, although in combined subjects there was a significant DRD2B X Area Interaction with glutamateNMDA receptor efficacy (F(1,57) 4.67; P 0.05), measured as the extent of glutamate-enhanced MK801 binding. In contrast, there was a significant Case-group X ADH3 X Area Interaction with glutamateNMDA receptor efficacy (F(3,57) 2.97; P 0.05). When GABAA receptor subunit isoform expression was examined, significant Case-group X Genotype X Area X Isoform interactions were found for EAAT2 with subunit mRNA (F(1,37) 4.22; P0.05), for GABB2 with isoform protein (F(1,37) 5.69; P 0.05), and for DRD2B with isoform protein (F(2,34)5.69; P0.05). The results suggest that subjects’ genetic makeup may modulate the effectiveness of amino acid-mediated transmission in different cortical regions, and thereby influence neuronal vulnerability to excitotoxicity.

Analysis of RNA-protein interactions by flow cytometry

Flow cytometry, in combination with advances in bead coding technologies, is maturing as a powerful high-throughput approach for analyzing molecular interactions. Applications of this technology include antibody assays and single nucleotide polymorphism mapping. This review describes the recent development of a microbead flow cytometric approach to analyze RNA-protein interactions and discusses emerging bead coding strategies that together will allow genome-wide identification of RNA-protein complexes. The microbead flow cytometric approach is flexible and provides new opportunities for functional genomic studies and small-molecule screening.

Probing the S100 protein family through genomic and functional analysis

The EF-hand superfamily of calcium binding proteins includes the S100, calcium binding protein, and troponin subfamilies. This study represents a genome, structure, and expression analysis of the S100 protein family, in mouse, human, and rat. We confirm the high level of conservation between mammalian sequences but show that four members, including S100A12, are present only in the human genome. We describe three new members of the S100 family in the three species and their locations within the S100 genomic clusters and propose a revised nomenclature and phylogenetic relationship between members of the EF-hand superfamily. Two of the three new genes were induced in bone-marrow-derived macrophages activated with bacterial lipopolysaccharide, suggesting a role in inflammation. Normal human and murine tissue distribution profiles indicate that some members of the family are expressed in a specific manner, whereas others are more ubiquitous. Structure-function analysis of the chemotactic properties of murine S100A8 and human S100A12, particularly within the active hinge domain, suggests that the human protein is the functional homolog of the murine protein. Strong similarities between the promoter regions of human S100A12 and murine S100A8 support this possibility. This study provides insights into the possible processes of evolution of the EF-hand protein superfamily. Evolution of the S100 proteins appears to have occurred in a modular fashion, also seen in other protein families such as the C2H2-type zinc-finger family. (C) 2004 Elsevier Inc. All rights reserved.

Construction of representative transcript and protein sets of human, mouse, and rat as a platform for their transcriptome and proteome analysis

The number of mammalian transcripts identified by full-length cDNA projects and genome sequencing projects is increasing remarkably. Clustering them into a strictly nonredundant and comprehensive set provides a platform for functional analysis of the transcriptome and proteome, but the quality of the clustering and predictive usefulness have previously required manual curation to identify truncated transcripts and inappropriate clustering of closely related sequences. A Representative Transcript and Protein Sets (RTPS) pipeline was previously designed to identify the nonredundant and comprehensive set of mouse transcripts based on clustering of a large mouse full-length cDNA set (FANTOM2). Here we propose an alternative method that is more robust, requires less manual curation, and is applicable to other organisms in addition to mouse. RTPSs of human, mouse, and rat have been produced by this method and used for validation. Their comprehensiveness and quality are discussed by comparison with other clustering approaches. The RTPSs are available at ftp://fantom2.gsc.riken.go.jp/RTPS/. (C). 2004 Elsevier Inc. All rights reserved.

Better prediction of protein contact number using a support vector regression analysis of amino acid sequence

Background: Protein tertiary structure can be partly characterized via each amino acid's contact number measuring how residues are spatially arranged. The contact number of a residue in a folded protein is a measure of its exposure to the local environment, and is defined as the number of C-beta atoms in other residues within a sphere around the C-beta atom of the residue of interest. Contact number is partly conserved between protein folds and thus is useful for protein fold and structure prediction. In turn, each residue's contact number can be partially predicted from primary amino acid sequence, assisting tertiary fold analysis from sequence data. In this study, we provide a more accurate contact number prediction method from protein primary sequence. Results: We predict contact number from protein sequence using a novel support vector regression algorithm. Using protein local sequences with multiple sequence alignments (PSI-BLAST profiles), we demonstrate a correlation coefficient between predicted and observed contact numbers of 0.70, which outperforms previously achieved accuracies. Including additional information about sequence weight and amino acid composition further improves prediction accuracies significantly with the correlation coefficient reaching 0.73. If residues are classified as being either contacted or non-contacted, the prediction accuracies are all greater than 77%, regardless of the choice of classification thresholds. Conclusion: The successful application of support vector regression to the prediction of protein contact number reported here, together with previous applications of this approach to the prediction of protein accessible surface area and B-factor profile, suggests that a support vector regression approach may be very useful for determining the structure-function relation between primary sequence and higher order consecutive protein structural and functional properties.

Genetic time-series analysis identifies a major QTL for in vivo alcohol metabolism not predicted by in vitro studies of structural protein polymorphism at the ADH1B or ADH1C loci

After ingestion of a standardized dose of ethanol, alcohol concentrations were assessed, over 3.5 hours from blood (six readings) and breath (10 readings) in a sample of 412 MZ and DZ twins who took part in an Alcohol Challenge Twin Study (ACTS). Nearly all participants were subsequently genotyped on two polymorphic SNPs in the ADH1B and ADH1C loci known to affect in vitro ADH activity. In the DZ pairs, 14 microsatellite markers covering a 20.5 cM region on chromosome 4 that includes the ADH gene family were assessed, Variation in the timed series of autocorrelated blood and breath alcohol readings was studied using a bivariate simplex design. The contribution of a quantitative trait locus (QTL) or QTL's linked to the ADH region was estimated via a mixture of likelihoods weighted by identity-by-descent probabilities. The effects of allelic substitution at the ADH1B and ADH1C loci were estimated in the means part of the model simultaneously with the effects sex and age. There was a major contribution to variance in alcohol metabolism due to a QTL which accounted for about 64% of the additive genetic covariation common to both blood and breath alcohol readings at the first time point. No effects of the ADH1B*47His or ADH1C*349Ile alleles on in vivo metabolism were observed, although these have been shown to have major effects in vitro. This implies that there is a major determinant of variation for in vivo alcohol metabolism in the ADH region that is not accounted for by these polymorphisms. Earlier analyses of these data suggested that alcohol metabolism is related to drinking behavior and imply that this QTL may be protective against alcohol dependence.

Shortcomings of protein removal prior to high performance liquid chromatographic analysis - A case study using method development for BAY 11-7082

During the analytical method development for BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile), using HPLC-MS-MS and HPLC-UV, we observed that the protein removal process (both ultrafiltration and precipitation method using organic solvents) prior to HPLC brought about a significant reduction in the concentration of this compound. The use of a structurally similar internal standard, BAY 11-7085 ((E)-3-[4-t-butylphenylsulfonyl]-2-propenenitrile), was not effective in compensating for the loss of analyte as the extent of reduction was different to that of the analyte. We present here a systematic investigation of this problem and a new validated method for the determination of BAY 11-7082. (c) 2006 Elsevier B.V. All rights reserved.

Leading coordinate analysis of reaction pathways in proton chain transfer: Application to a two-proton transfer model for the green fluorescent protein

The ‘leading coordinate’ approach to computing an approximate reaction pathway, with subsequent determination of the true minimum energy profile, is applied to a two-proton chain transfer model based on the chromophore and its surrounding moieties within the green fluorescent protein (GFP). Using an ab initio quantum chemical method, a number of different relaxed energy profiles are found for several plausible guesses at leading coordinates. The results obtained for different trial leading coordinates are rationalized through the calculation of a two-dimensional relaxed potential energy surface (PES) for the system. Analysis of the 2-D relaxed PES reveals that two of the trial pathways are entirely spurious, while two others contain useful information and can be used to furnish starting points for successful saddle-point searches. Implications for selection of trial leading coordinates in this class of proton chain transfer reactions are discussed, and a simple diagnostic function is proposed for revealing whether or not a relaxed pathway based on a trial leading coordinate is likely to furnish useful information.

Pattern analysis on protein properties of Chinese and CIMMYT spring wheat cultivars sown in China and CIMMYT

Improvement of processing quality is a very important objective for Chinese wheat breeding programs. Twenty-five CIMMYT and Chinese spring wheat cultivars were grown at four managed conditions by CIMMYT in Cd. Obregon, Sonora, Mexico and in nine environments in China, over two successive wheat seasons from 2000 to 2002. These trials were used to identify patterns of cultivar, environment and cultivar x environment interactions, and to determine opportunities for indirect selection for protein content and the protein-quality related parameter, SDS sedimentation (SDSS) value. The cultivar Inqalab 91 showed low levels of interaction with environments in the 2000-01 crop cycle for protein content, and expressed intermediate levels for both protein content and SDSS value, across most of the environments in both years. Longmai 26 had consistently high protein content and SDSS value across environments in both years, indicating that it is possible to breed cultivars expressing high yields with good protein properties. Cluster analyses revealed that cultivars grouped differently for protein content and SDSS value. Besides photoperiod, water availability appeared to influence the ranking of cultivars for protein content and SDSS value. Temperature and soil type may underlie the observed interactions for protein content, while temperature may also be a factor associated with interactions for SDSS value. The full irrigation managed environment in Mexico, with the cultivars sown on raised beds two months later than optimum and exposing them to late heat, clustered together with the Chinese environments Huhhot, Yongning, and Hejin in the 2000-01 season for SDSS value. This indicates that there is an opportunity to exploit indirect responses to selection in the CIMMYT management environments for SDSS value with relevance for China's spring wheat regions. However, there seemed little chance for positive indirect selection in CIMMYT's managed environments for China in regard to protein content, as environments clustered distinctly. Pattern analyses permitted a sensible and useful summary for this multi environment experiment, helping in understanding natural relationships and variations in cultivar performance among the various environment groups, and assisting in the structuring of environments.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

Genomic and proteomic analysis of synaptic protein expression in the brains of human alcoholics

Alcoholism results in changes in the human brain which reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of mRNA and proteins in key cells and brain areas. Long-term alcohol abuse also results in damage to selected regions of the cortex. We have used cDNA microarrays to show that less than 1% of mRNA transcripts differ signifi cantly between cases and controls in the susceptible area and that the expression profi le of a subset of these transcripts is suffi cient to distinguish alcohol abusers from controls. In addition, we have utilized a 2D gel proteomics based approach to determine the identity of proteins in the superior frontal cortex (SFC) of the human brain that show differential expression in controls and long term alcohol abusers. Overall, 182 proteins differed by the criterion of > 2-fold between case and control samples. Of these, 139 showed signifi cantly lower expression in alcoholics, 35 showed signifi cantly higher expression, and 8 were new or had disappeared. To date 63 proteins have been identifi ed. The expression of one family of proteins, the synucleins, has been further characterized using Real Time PCR and Western Blotting. The expression of alpha-synuclein mRNA was signifi cantly lower in the SFC of alcoholics compared with the same area in controls (P = 0.01) whereas no such difference in expression was found in the motor cortex. The expression of beta- and gamma- synuclein were not signifi cantly different between alcoholics and controls. In contrast, the pattern of alphasynuclein protein expression differs from that of the corresponding RNA transcript. Because of the key role of synaptic proteins in the pathogenesis of alcoholism, we are developing 2-D DIGE based techniques to quantify expression changes in synaptosomes prepared from the SFC of controls and alcoholics.