Lung proteome alterations in a mouse model for nonallergic asthma

A mouse model for nonatopic asthma was employed to study the alterations of the lung proteome to gain insight into the underlying molecular mechanisms of disease pathophysiology post-challenge. Lung samples from asthmatic and control mice were used to generate 24 high quality two-dimensional electrophoresis gels wherein 2115 proteins were examined for disease relevance. In total, 23 proteins were significantly up- or down-regulated following hapten-challenge of dinitro-fluorobenzene-hypersensitive mice. Twenty proteins were identified by mass spectrometry, of which 18 could be linked to asthma related symptoms, such as stress and inflammation, lung detoxification, plasma exudation and/or tissue remodeling. As such, proteomics was clearly vindicated as a means of studying this complex disease phenomenon. The proteins found in this study may not necessarily play a role in the immunological mechanisms and/or pathophysiology of asthma development. However, they may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy. The mathematics of achieving statistical confidence from low numbers of gel replicates containing large numbers of independent variables stress the need for high numbers of replicates to better sample the population of proteins revealed by two-dimensional gel electrophoresis.     

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.     

A mouse to human search for plasma proteome changes associated with pancreatic tumor development

Background

The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer.

Methods and Findings

Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer.

Conclusions

Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection.     

Mass spectrometry-based detection and quantification of plasma glycoproteins using selective reaction monitoring

Mass spectrometry-based targeted proteomics is a rapidly expanding method for quantifying proteins in complex clinical samples such as plasma. In conjunction with the stable isotope dilution method, selected reaction monitoring (SRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRM assays is the reduction of interference by lowering the background. This can be achieved by the selective isolation of a subproteome, such as N-glycosylated proteins, from the original sample. The present protocol includes the development and optimization of SRM assays associated with each peptide of interest and the qualification of assays in the biological matrix to establish the limits of detection and quantification. The protocol also describes the enrichment of formerly N-glycosylated peptides relying on periodate oxidation of glycan moieties attached to the proteins, their immobilization on solid supports through hydrazide chemistry, proteolysis and enzymatic release of the formerly N-glycosylated peptides.     

Early dysregulation of the mitochondrial proteome in a mouse model of Alzheimer's disease

Mitochondrial structural and functional alterations appear to play to an important role in the pathogenesis of Alzheimer's disease (AD). In the present study, we used a quantitative comparative proteomic profiling approach to analyze changes in the mitochondrial proteome in AD. A triple transgenic mouse model of AD (3xTg-AD) which harbors mutations in three human transgenes, APP(Swe), PS1(M146V) and Tau(P301L), was used in these experiments. Quantitative differences in the mitochondrial proteome between the cerebral cortices of 6-month-old male 3xTg-AD and non-transgenic mice were determined by using two-dimensional difference gel electrophoresis (2D-DIGE) and tandem mass spectrometry. We identified 23 different proteins whose expression levels differed significantly between triple transgenic and non-transgenic mitochondria. Both down-regulated and up-regulated mitochondrial proteins were observed in transgenic AD cortices. Proteins which were dysregulated in 3xTg-AD cortices functioned in a wide variety of metabolic pathways, including the citric acid cycle, oxidative phosphorylation, pyruvate metabolism, glycolysis, oxidative stress, fatty acid oxidation, ketone body metabolism, ion transport, apoptosis, and mitochondrial protein synthesis. These alterations in the mitochondrial proteome of the cerebral cortices of triple transgenic AD mice occurred before the development of significant amyloid plaque and neurofibrillary tangles, indicating that mitochondrial dysregulation is an early event in AD.     

Immunoproteomics: Mass spectrometry-based methods to study the targets of the immune response

The mammalian immune system has evolved to display fragments of protein antigens derived from microbial pathogens to immune effector cells. These fragments are typically peptides liberated from the intact antigens through distinct proteolytic mechanisms that are subsequently transported to the cell surface bound to chaperone-like receptors known as major histocompatibility complex (MHC) molecules. These complexes are then scrutinized by effector T cells that express clonally distributed T cell receptors with specificity for specific MHC-peptide complexes. In normal uninfected cells, this process of antigen processing and presentation occurs continuously, with the resultant array of self-antigen-derived peptides displayed on the surface of these cells. Changes in this peptide landscape of cells act to alert immune effector cells to changes in the intracellular environment that may be associated with infection, malignant transformation, or other abnormal cellular processes, resulting in a cascade of events that result in their elimination. Because peptides play such a crucial role in informing the immune system of infection with viral or microbial pathogens and the transformation of cells in malignancy, the tools of proteomics, in particular mass spectrometry, are ideally suited to study these immune responses at a molecular level. Here we review recent advances in the studies of immune responses that have utilized mass spectrometry and associated technologies, with specific examples from collaboration between our laboratories.     

Use of a mouse chimaeric model to study cell migration patterns in the small intestinal epithelium

The pattern of cells migration in the small intestinal epithelia of a RIII/ro----C57BL/6J mouse aggregation chimaera is demonstrated using Dolichos biflorus agglutinin-peroxidase (DBA) conjugate as a strain-specific marker. Using serial tangential sections of heterogeneously stained villi and plotting the distribution of labelled/unlabelled cells with a drawing tube, and by three-dimensional reconstruction with the aid of computer graphics, we show the migration pathway to be in tight cohorts of similar DBA-peroxidase staining type, which move upwards in straight lines. There is little cell mixing either on the villus or along the crypt-villus junctions. Our observations also show for the first time that a single crypt can feed cells to more than one villus. They also suggest that either cell loss is not confined to the villus tips but can take place from the villus sides, or that there is marked asynchrony of cell production between crypts.     

Enhanced analysis of the mouse plasma proteome using cysteine-containing tryptic glycopeptides

A comprehensive understanding of the mouse plasma proteome is important for studies using mouse models to identify protein markers of human disease. To enhance our analysis of the mouse plasma proteome, we have developed a method for isolating low-abundance proteins using a cysteine-containing glycopeptide strategy. This method involves two orthogonal affinity capture steps. First, glycoproteins are coupled to an azlactone copolymer gel using hydrazide chemistry and cysteine residues are then biotinylated. After trypsinization and extensive washing, tethered N-glycosylated tryptic peptides are released from the gel using PNGase F. Biotinylated cysteinyl-containing glycopeptides are then affinity selected using a monomeric avidin gel and analyzed by LC-MS/MS. We have applied the method to a proteome analysis of mouse plasma. In two independent analyses using 200 muL each of C57BL mouse plasma, 51 proteins were detected. Only 42 proteins were seen when the same plasma sample was analyzed by glycopeptides only. A total of 104 N-glycosylation sites were identified. Of these, 17 sites have hitherto not been annotated in the Swiss-Prot database whereas 48 were considered probable, potential, or by similarity - i.e., based on little or no experimental evidence. We show that analysis by cysteine-containing glycopeptides allows detection of low-abundance proteins such as the epidermal growth factor receptor, the Vitamin K-dependent protein Z, the hepatocyte growth factor activator, and the lymphatic endothelium-specific hyaluronan receptor as these proteins were not detected in the glycopeptide control analysis.     

Age-related proteome analysis of the mouse brain: a 2-DE study

2-DE remains the most popular and versatile protein separation method among a rapidly growing array of various proteomics technologies. However, variability in sample processing, experimental design and data analyses results in a limited cross-validation between studies performed in different laboratories. One of the goals of the Human Proteome Organization (HUPO) is to establish standards and guidelines for proteomics studies. We contributed to the HUPO Brain Proteome Project by analyzing brains from neonatal and adult mice using 2-DE. Here we propose a standard workflow to analyze 2-DE images and extract statistically significant differences. After differential analysis and identification by MALDI-TOF/TOF, dihydropyrimidinase-related proteins, brain FABP, stathmin, isocitrate dehydrogenase, gamma enolase, annexin V, glutamine synthetase, creatine kinase B chain, triosephosphate dehydrogenase, and malate dehydrogenase were found differentially expressed between the two groups. The functions and potential mechanisms underlying the variation observed for these proteins are discussed.     

Mass spectrometry-based immuno-precipitation proteomics - the user's guide

Immuno-precipitation (IP) experiments using MS provide a sensitive and accurate way of characterising protein complexes and their response to regulatory mechanisms. Differences in stoichiometry can be determined as well as the reliable identification of specific binding partners. The quality control of IP and protein interaction studies has its basis in the biology that is being observed. Is that unusual protein identification a genuine novelty, or an experimental irregularity? Antibodies and the solid matrices used in these techniques isolate not only the target protein and its specific interaction partners but also many non-specific 'contaminants' requiring a structured analysis strategy. These methodological developments and the speed and accuracy of MS machines, which has been increasing consistently in the last 5 years, have expanded the number of proteins identified and complexity of analysis. The European Science Foundation's Frontiers in Functional Genomics programme 'Quality Control in Proteomics' Workshop provided a forum for disseminating knowledge and experience on this subject. Our aim in this technical brief is to outline clearly, for the scientists wanting to carry out this kind of experiment, and recommend what, in our experience, are the best potential ways to design an IP experiment, to help identify possible pitfalls, discuss important controls and outline how to manage and analyse the large amount of data generated. Detailed experimental methodologies have been referenced but not described in the form of protocols.     

Evaluation of strategy for analyzing mouse liver plasma membrane proteome

Plasma membrane (PM) proteome is one of the major subproteomes present in the cell,and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.     

Mass spectrometry-based proteomics for the detection of plant pathogens

Plant diseases caused by fungi, oomycetes, viruses, and bacteria are devastating both to the economy and to the food supply of a nation. Therefore, the development of new, rapid methods to identify these pathogens is a highly important area of research that is of international concern. MS-based proteomics has become a powerful and increasingly popular approach to not only identify these pathogens, but also to better understand their biology. However, there is a distinction between identifying a pathogen protein and identifying a pathogen based upon the detection of one of its proteins and this must be considered before the general application of MS for plant pathogen detection is made. There has been a recent push in the proteomics community to make data from large-scale proteomics experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MS as a universal plant pathogen detection technology.     

Mass spectrometry-based proteomics for translational research: a technical overview

Mass spectrometry-based investigation of clinical samples enables the high-throughput identification of protein biomarkers. We provide an overview of mass spectrometry-based proteomic techniques that are applicable to the investigation of clinical samples. We address sample collection, protein extraction and fractionation, mass spectrometry modalities, and quantitative proteomics. Finally, we examine the limitations and further potential of such technologies. Liquid chromatography fractionation coupled with tandem mass spectrometry is well suited to handle mixtures of hundreds or thousands of proteins. Mass spectrometry-based proteome elucidation can reveal potential biomarkers and aid in the development of hypotheses for downstream investigation of the molecular mechanisms of disease.     

Integrated pipeline for mass spectrometry-based discovery and confirmation of biomarkers demonstrated in a mouse model of breast cancer

Despite their potential to impact diagnosis and treatment of cancer, few protein biomarkers are in clinical use. Biomarker discovery is plagued with difficulties ranging from technological (inability to globally interrogate proteomes) to biological (genetic and environmental differences among patients and their tumors). We urgently need paradigms for biomarker discovery. To minimize biological variation and facilitate testing of proteomic approaches, we employed a mouse model of breast cancer. Specifically, we performed LC-MS/MS of tumor and normal mammary tissue from a conditional HER2/Neu-driven mouse model of breast cancer, identifying 6758 peptides representing >700 proteins. We developed a novel statistical approach (SASPECT) for prioritizing proteins differentially represented in LC-MS/MS datasets and identified proteins over- or under-represented in tumors. Using a combination of antibody-based approaches and multiple reaction monitoring-mass spectrometry (MRM-MS), we confirmed the overproduction of multiple proteins at the tissue level, identified fibulin-2 as a plasma biomarker, and extensively characterized osteopontin as a plasma biomarker capable of early disease detection in the mouse. Our results show that a staged pipeline employing shotgun-based comparative proteomics for biomarker discovery and multiple reaction monitoring for confirmation of biomarker candidates is capable of finding novel tissue and plasma biomarkers in a mouse model of breast cancer. Furthermore, the approach can be extended to find biomarkers relevant to human disease.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.     

Synaptic proteome changes in a DNA repair deficient ercc1 mouse model of accelerated aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged Ercc1 mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and Ercc1 mutant hippocampal neurons displayed normal outgrowth and synapse formation in vitro. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline.