Mass spectrometric analysis if peptides and proteins requires samples as pure as possible. Salts and small molecules can significantly disturb the analysis. Additionally, suppression effects can occur possibly leading to a restraint of the peptide or protein of interest. A fractionation if the peptide/protein mixture may therefore be advantageous. Novel ClinProtTM beads (figure.1) are magntic microparticles available with different surface functionalities. They were developed to be used for peptide profiling in clinical proteomics applications. Here, we show their utilization for low abundant peptide and protein (phosphorylated Peptides and glycopeptides )enrichment, fractionation and purification prior to MALDI-TOF MS at some selected applications.

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In the present time, human liver proteins identified by combination of detergent-solubilization, 2-DE and MALDI/TOF MS are less than expected estimation from the genome database. For identification of the causative abnormality of the protein functions in the intractable disease more comprehensive and precise informations are required on each component proteins constructing the signal road from the receptors outside of the cell membrane to nucei or other organelles. To resolve and identify as many as possible proteins from the human liver specimen, the homogenate of the specimen was fractionated to the cytosol, ER, mitochondria, and, nuclei and cell debris fractions. The proteins in each fractions were solubilized with different solvent systems. Various kinds of protein not extracted from the whole homogenate were identified by the extraction after the fractionation.

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We previously identified that two genetic elements, ASE and AIE, are responsible for ASE/AIE-mediated age-related regulatory mechanism of gene expression through extensive studies of transgenic mice carrying minigenes of human blood coagulation factor IX (J Thromb Haemost 3, 909-914, 2005). To gain understanding how liver proteins are regulated for their expression throughout the lifespan, we carried out global analyses of age-related changes of mouse liver nuclear proteins. To gain understanding how liver proteins are regulated for their expression throughout the lifespan, we carried out global analyses of age-related changes of mouse liver nuclear proteins. Liver nuclear proteins prepared from mice (C57BL/6xSJL, male) at 1, 3, 6, 12, 18 and 21 months of age (n=10-20/age point) were extensively analyzed by 2-dimensional gel electrophoresis (2DGE) (pH range 4-11) and MALDI-TOF/MS. Out of more than 6000 protein spots separated by 2DGE, about 4500 protein spots were selected for analyses with MALDI-TOF/MS and Mascot Protein Identifier program. By removing duplications, over 2500 protein spots including isomer spots were found unique, and subjected to quantitative analyses with the PDQuest and GeneSpring programs. Among many complex age-related expression profiles, at least six unique and fundamental age-related profiles, including puberty-onset increase or decrease and old-age associated increase or decrease, were observed. Specific age-related changes in expression profiles, likely due to changes in post-translational modifications, were observed for some isomers. These findings support that there exist multiple fundamental age-axis regulatory mechanisms for liver nuclear proteins. This is the first comprehensive and quantitative analyses of liver nuclear protein expressions in relation to age and this research will facilitate various basic studies related to epigenetics and age-related diseases as well as industrial applications for drug discovery.

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NMR based metabolic profiling is powerful and useful tool for evaluating phenotypes as whole metabolite 1D NMR spectrum using biofluids. We tested this method on newborn infants' urine samples including normal and urine diseases. NMR data were directly processed by developing version of Alice2 for Metabolome program package (JEOL Datum). PCA and SIMCA(Soft Independent Modeling of Class Analogy) method clearly discriminated disease samples from healthy. Additionally, we found the potential to evaluate remission status in treated babies by this method.

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NMR based metabolic profiling is a powerful tool for evaluating phenotypes as whole metabolite 1D NMR spectrum using biofluids. We tested this method on rat urine samples from control and streptozotocin-induced diabetes rats. NMR data were directly processed by 'AlICE2 for Metabolome' program package (JEOL). PCA and SIMCA (Soft Independent Modeling of Class Analogy) method clearly discriminated dosed-rat samples from control, including time course or individual differences.

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Hepatic fibrosis is an important consequence of inflammatory disorders in the liver with hepatitis C virus, and ultimately progresses to cirrhosis and hepatocelluler carcinoma. Diagnosis at the early stage of chronic hepatitis C and monitor the progress to cirrhosis is important for the liver cancer prevention. Here, we established comprehensive and quantitative MALDI-TOF mass spectrometry-based serum peptide profiles for biomarker discovery in the liver diseases. Peptide profiles were obtained from 2D-µHPLC MALDI-TOF MS of low molecular weight protein fraction in serum from hepatitis/cirrhosis patients (each 20 cases) and 20 normal controls. Peptides specifically observed in the diseases were found on the differential analysis software, DeView, originally developed by us. The peptides from each sample were mapped in 2-dimension consist of m/z and HPLC fraction, and intensity of signals were compared among samples. These potential biomarkers for inflammation and fibrosis were identified by MS/MS or MSⁿ analysis on MALDI TOF-TOF MS or MALDI-QIT-TOF MS. As a result, 193 peptides showed 0.8 or more value in Area Under Curve (AUC) of Receiver Operating Characteristic (ROC). Among them, 72 peptides were unique in the chronic hepatitis.

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Plasma protein proteome by 2D separation combined with the LC-MS is the main trend to search the disease marker proteins. To increase the efficiency of the process, abundant proteins partitioning is also noteworthy. We compared the human plasma protein profiles before and after the meal by 2-dimensional LC liquid separation technique (ProteomeLab PF2D) after abundant proteins partitioning by IgY column (ProteomeLab IgY), and found out the many differences. Then some differences were analyzed by LC-ESI-Q-TOF (Micro Q-TOF) after trypsin digestion, and identified some proteins. We also discuss the efficiency of the lysine residues isotope labeling technique preceding the liquid separation process.

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Superconducting detectors have unconventional performance as detectors for accelerated molecules. It is theoretically predicted that the superconducting detectors have a detection efficiency of 100% and enable measurement of kinetic energies of molecules. These functions are especially important for TOF MS. We have experimentally confirmed that the 100% detection efficiency is realized up to 1 MDa in MALDI-TOF MS. The perfect detection efficiency may play an important role in biostandard or synthetic polymer standard. Furthermore, the kinetic energy measurement may initiate new fragmentation analysis not only for ions, but also neutral fragments. In this talk, superconducting detectors are introduced as a tool to optimize ionization condition and to analyze fragmentation. We are now developing one-hundred pixel array superconducting detectors for quantitative and structural analysis.

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Proteomics research is very useful throughout the entire research and development process of new drugs. Novel protein profiling technologies and platforms have increased the possibilities of identifying new drug targets and biomarkers, thus making them an important tool for developing more effective and safer drugs. Pharma and biotech companies are increasingly seeking to include biomarker information with every new CD delivered from the discovery departments. The sequencing,- and structure elucidating- capabilities by the new generation of mass spectrometers has made it possible to identify protein entities at both medium and low abundant expression regions . With respect to proteomic patterns and annotations identified from clinical samples offers a new dimension to clinical trials to evaluate the effects of drug treatment which also correlate to the mechanisms of drug action. Protein biomarkers can assist in diagnosing a disease at the biochemical level or discriminating the responses of different patients to the same medical treatment. A combination of proteomic strategies, including high resolution nano-capillary chromatography, 2-dimensinal gel electrophoresis, interfaced to mass spectrometry as well as protein microchip arrays has been recognised as important tools in biomarker discovery. Within AstraZeneca there is currently a major emphasis on the study of proteins and peptides from basic discovery to clinical development, with a global organisation to support it within the respective research and disease areas.. We have developed technology platforms that can identify biomarker candidates in a number of biofluids as well as tissue and cells.. Outlines and perspectives will be presented and discussed, exemplified by proteomics studies within various therapeutic areas of interest to AstraZeneca.

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Neural stem cells are capable of proliferation and differentiation into neurons and glial cells. With use of their property, replacement of lost neurons by their transplantation constitutes a promising new approach to treatment of progressive neurodegenerative disease. The protein expression in neural stem cells differentiated from embryonic stem (ES) cells has not been well characterized. We investigated protein profile of mouse neural stem cells differentiated from ES cells into neurons. The neural cells were produced from ES cells by Neural Stem Sphere (NSS) method. The NSS with a periphery of neural stem cells is formed from a colony of ES cells by culture in astrocyte-conditioned medium under free-floating conditions. Culturing the NSSs on an adhesive substrate with FGF-2 promoted migration of the neural stem cells onto the substrate. The neural stem cells were harvested, expanded, and differentiated exclusively into neurons. We ascertained by real-time RT-PCR analysis that nestin was up-regulated in neural stem cells, and down-regulated in ES cells and neurons, and that NF-M and MAP2 were up-regulated in neurons. Lysates of the cells in each stage were subjected to two-dimensional gel electrophoresis, and proteins showing the changes in their expression levels were detected. These proteins were further investigated by peptide mass fingerprinting. We identified vimentin, creatine kinase B, thioredoxin 1 and some proteins with unknown biological functions in the database, which were up-regulated in neural stem cells, and down-regulated in ES cells and neuron. Real-time RT-PCR analysis indicated that mRNA levels of creatine kinase B and thioredoxin 1 were up-regulated in neural stem cells and neurons. These results suggest that ES cells are reorganized upon their differentiation into neuronal cells via neural stem cells, and suggest the possibility that post-translational modifications of the proteins are processing during their differentiation into neurons.

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In an attempt to disclose phosphoproteome of the normal rat glomerulus, which is assumed to play critical roles in the physiology and pathophysiology of the kidney, we have conducted a proteomic study by combining 1-DE or 2-DE separation and immunochemical analyses using anti-phosphotyrosine antibodies in order to identify tyrosine-phosphorylated proteins in the normal rat glomerulus. Western blotting analysis revealed the presence of tyrosine-phosphorylated proteins of 62, 70, 120, 145 ,200, 220-kDa, of which those of 62, 70, 120, 145, 220-kDa were predominantly detected in the glomerulus, while others were commonly present in the three compartments; glomerulus, cortex, and medulla. These phosphorylation was augmented by orthovanadate, and abolished in the presence of phosphotyrosine but not of phoshoserine or phosphothreonine. Immunofluorescence microscopy indicated the predominant localization of phosphotyrosine staining along capillary walls in the glomerulus with less intense staining along the tubular epithelium. Immunoelectron microscopy also indicated the predominant localization of phosphotyrosin immunoreactivity to the basal membrane of glomerular epithelial cell (podocyte) foot processes adjacent to GBM and the basement of slit diaphragm. With MALDI-TOF MS and LC-MS/MS analysis, we have so far identified 4 tyrosine-phosphorylated proteins as talin (220-kDa), vinculin (120-kDa), ezrin (74-kDa), Hsc70 (70-kDa). Immunoblotting analysis using antibodies against talin, vinculin and Hsc70 revealed their ubiquitous expression in all the three compartments of kidney. These results suggest that talin, vinculin and ezrin (membrane cytoskeleton-associated proteins), and Hsc70 (a constitutive member of Hsp 70 family) are phosphorylated almost exclusively in the glomerulus, presumably in podocytes, highly specialized epithelial cells structurally adapted to facilitate bulk flow of the glomerular filtrate through their intercellular structures, slit diaphragms.

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SAMP8 mice exhibit age-related deficits in learning and memory at earlier stages of the life span than SAMR1 mice. It has been reported that SAMP8 mice show a higher oxidative status in the brain. Using proteomics tools, we compared oxidative damage to proteins and alteration in the protein expression in the brain between SAMP8 and SAMR1 mice at the age of 7 months. We analyzed the protein expressions in SAMP8 and SAMR1 brains using 2D-DIGE. After DIGE analysis, these differentiated proteins were identified by PMF analysis with MALDI-ToF MS. Three proteins were down-regulated, whereas two proteins were up-regulated in SAMP8 compared with SAMR1. We also demonstrated that the total amount of carbonylated proteins in the hippocampus was increased in SAMP8 compared with SAMR1. The carbonylation of actin, glial fibrillary acidic protein and mu-crystallin homolog was especially elevated in SAMP8. These proteins may be valuable candidates for biomarkers of aging.

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Neurons, oligodendrocytes and astrocytes are differentiated and maturated from their neural stem cells through immature progenitor cells. Many researchers have attempted to identify specific marker proteins, since the functional differences among those neural cell types should be come from the difference in protein expression in the differentiated cells. However, most of the differentiation marker proteins were identified by immunocytochemical staining using specific antibodies and confirmed by RT-PCR in their message level. Therefore many valuable marker proteins have not yet been cleared completely. The most significant reason for the lack in information about the neural cell type-specific protein biomarkers might be the difficulty in preparation of each neural cell type from mature brain tissues for the proteomic analysis. So we tried to isolate immature neural progenitor cells from rat embryonic brain tissues and differentiate into neurons, oligodendrocytes and astrocytes for the differential proteome analysis. As the result of the comparative research, we detected 788 spots in neurons, 695 spots in oligogendrocytes, 633 spots in astrocyts, 691 spots in immature progenitor cells, and 747 spots in its conditioned medium. However, only 107 spots were expressed commonly in all cell types. The expression levels of 14-3-3 protein isoforms and brain lipid-binding protein were found significantly lower in astrocytes than neurons and oligodendrocytes.

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RIKEN Structural Genomics/Proteomics Initiative (RSGI) (http://www.rsgi.riken.jp) was organized by RIKEN Genomics Sciences Center and Harima Institute at SPring-8 in 2001 to conduct genome- and proteome-based structural biology (structural genomics and proteomics). RSGI has been integrated into the National Project on Protein Structural and Functional Analyses ("NPPSFA" or "Protein 3000"), organized by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), as one center of the program for comprehensive studies.
We aims to study both structures and molecular functions of eukaryotic protein families involved in phenomena of biological and medical importance. Domain(s) belonging to selected families are screened in terms of the suitability for the structure determination. In the screening stage, we use the automated protein production based on a high-yield cell-free synthesis, and the NMR spectroscopy. The winners in the screening stage are subjected to larger-scale cell-free production of the uniformly ¹³C/¹⁵N-labeled samples for the NMR spectroscopy, and then step to structure determination. A part of the samples that are judged to be more suitable for the X-ray crystallography are prepared in the selenomethionine-substituted form for structure determination by MAD phasing. To accelerate the NMR analysis, we have developed a software package, KUJIRA, for the systematic and interactive NMR data analysis, and the program CYANA for automated structure calculation.
We determined more than 900 structures by NMR spectroscopy according to this workflow.

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[Introduction] Although ejaculated mammalian sperm can not fertile the oocyte, capacitated mammalian sperm can do. Capacitation is a qualitative change of mammalian spermatozoa where capacitated spermatozoa are acrosome reacted. After the acrosome reaction, they are then hyperactivated. The acrosome reaction is a specialized form of exocytosis involving the fusion of the outer acrosomal membrane with the sperm plasma membrane overlying the acrosome, followed by the vesiculation of fused membranes and release of acrosomal contents. In contrast, hyperactivation is a specialized movement of sperm flagellum involving a propulsive force in order to pass through the zona pellucida. It has been demonstrated that many sperm proteins are phosphorylated during capacitation and hyperactivation. Progesterone functions to stimulate both capacitation and the acrosome reaction of mammalian sperm. Although progesterone controls cell functions through genomic pathways such as gene expression on many types of cells, it causes acrosome reaction by a non-genomic pathway on sperm. It is not always clear, however, whether progesterone affects hyperactivation of sperm. In the present study we examined whether progesterone affected hyperactivation using hamster sperm. [Results and conclusion] Progesterone caused a dose-dependent acceleration in sperm hyperactivation until 20ng/ml, although acceleration of hyperactivation was inhibited above this concentration. Acceleration of hyperactivation by progesterone was inhibited by a progesterone receptor antagonist. Moreover, progesterone bound to sperm head cell membranes. It has been suggested that tyrosine phosphorylation is closely associated with sperm capacitation. Progesterone also accelerated tyrosine phosphorylation. From these results it is likely that progesterone regulates sperm hyperactivation by non-genomic pathways with tyrosine phosphorylation via progesterone receptors on the sperm head cell membrane.

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Advances in LC/MS technology have opened the doors for protein biomarker discovery. However, successful biomarker discovery largely depends on the ability to interpret large volumes of data. This presentation discusses the Rosetta Elucidator system, designed for managing complex proteomics data and identifying differentially expressed proteins across multiple treatment groups or disease states, and how it enables protein biomarker discovery. We will discuss the application of the system to HIV biomarker discovery.
HIV–1 infection remains a therapeutically challenge due to frequent viral gene mutations. As HIV–1 proteins have been shown to co–opt host machinery for viral replication, countering alterations in host gene expression induced by viral infection may help interfere with viral replication. We describe a characterization of time–course HIV–1 infection using microarrays and label–free LCMS, along with functional characterization of cellular pathways exhibiting various extent of accord between mRNA and protein levels.
Rosetta Elucidator System was used to align LC retention time, quantify time–aligned isotopic clusters, and identify proteins with ProteinProphets. Initial analysis yielded 116 proteins exhibiting changes in abundance upon HIV–1 infection related to uninfected cells. Data–dependent analysis has so far returned ∼400 protein ID’s from our CD4+ T cells. To isolate proteins exhibiting such differential expression in a kinetic manner, Two–way ANOVA was performed on the relative protein abundance ratios from the start and end of active virus production. Pathway analysis on the resulting list of 44 proteins revealed perturbation in proteolysis, apoptosis, and cell cycling.
Our integrative approach has shown the effects on host mRNA and protein expression and cellular functions over the course of HIV–1 replication.

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