Targeted proteomics using immunoblotting technique for studying dysregulation of ion transporters in renal disorders

Renal salt and water transport physiology has benefited tremendously from the rapid advance of proteomics. Proteomics developed as a fast-throughput means of screening for global changes in proteins in a selected tissue, organ or cell type, as a logical offshoot of similar comprehensive, messenger RNA array-type technology. Targeted proteomics utilizes similar techniques but examines a predetermined set of proteins. One approach that has been rigorously employed over the last 10 years to evaluate differences in renal protein abundances due to a treatment or genotype has been parallel semiquantitative immunoblotting using antibody arrays. This approach, and newer ones on the horizon, provide a rapid global overview of regulation of the individual proteins whose integrated action determines overall renal sodium or water reabsorption.     

Electrophoresis in 2004: The Annual Meeting of the American Electrophoresis Society

Protein phosphorylation plays a critical role in the signaling pathways regulating water and solute transport in the distal renal tubule (i.e., renal collecting duct). A central mediator in this process is the antidiuretic peptide hormone arginine vasopressin, which regulates a number of transport proteins including water channel aquaporin-2 and urea transporters (UT-A1 and UT-A3). Within the past few years, tandem mass spectrometry-based proteomics has played a pivotal role in revealing global changes in the phosphoproteome in response to vasopressin signaling in the renal collecting duct. This type of large-scale ‘shotgun’ approach has resulted in an exponential increase in the number of phosphoproteins known to be regulated by vasopressin and has expanded on the established signaling mechanisms and kinase pathways regulating collecting duct physiology. This article will provide a brief background on vasopressin action, will highlight a number of recent quantitative phosphoproteomic studies in both native rat kidney and cultured collecting duct cells, and will conclude with a perspective focused on emerging trends in the field of phosphoproteomics.     

Phosphoproteomics of vasopressin signaling in the kidney

Protein phosphorylation plays a critical role in the signaling pathways regulating water and solute transport in the distal renal tubule (i.e., renal collecting duct). A central mediator in this process is the antidiuretic peptide hormone arginine vasopressin, which regulates a number of transport proteins including water channel aquaporin-2 and urea transporters (UT-A1 and UT-A3). Within the past few years, tandem mass spectrometry-based proteomics has played a pivotal role in revealing global changes in the phosphoproteome in response to vasopressin signaling in the renal collecting duct. This type of large-scale 'shotgun' approach has resulted in an exponential increase in the number of phosphoproteins known to be regulated by vasopressin and has expanded on the established signaling mechanisms and kinase pathways regulating collecting duct physiology. This article will provide a brief background on vasopressin action, will highlight a number of recent quantitative phosphoproteomic studies in both native rat kidney and cultured collecting duct cells, and will conclude with a perspective focused on emerging trends in the field of phosphoproteomics.     

Characterization and quantitation of membrane proteomes using multidimensional MS-based proteomic technologies

A major goal of proteomics is to develop methods that enable the systematic characterization of every protein within the cell or particular subcellular proteome using a single analytical platform. Although the equivalent has already been achieved in genomics, reaching this goal in proteomics represents a much greater challenge due to the wide dynamic range of protein expression, numerous post-translational modifications and remarkable physicochemical heterogeneity of proteins. A major analytical challenge has involved developing more effective means for proteome-scale investigations of membrane proteins, whose solubility differs drastically from that of cytoplasmic proteins. Fortunately, rapid progress has increased the ability to characterize this critically important class of proteins on a scale analogous to that of aqueous soluble proteins.     

Characterization and quantitation of membrane proteomes using multidimensional MS-based proteomic technologies

A major goal of proteomics is to develop methods that enable the systematic characterization of every protein within the cell or particular subcellular proteome using a single analytical platform. Although the equivalent has already been achieved in genomics, reaching this goal in proteomics represents a much greater challenge due to the wide dynamic range of protein expression, numerous post-translational modifications and remarkable physicochemical heterogeneity of proteins. A major analytical challenge has involved developing more effective means for proteome-scale investigations of membrane proteins, whose solubility differs drastically from that of cytoplasmic proteins. Fortunately, rapid progress has increased the ability to characterize this critically important class of proteins on a scale analogous to that of aqueous soluble proteins.     

Computational methods for the comparative quantification of proteins in label-free LCn-MS experiments

Liquid chromatography (LC) coupled to electrospray mass spectrometry (MS) is well established in high-throughput proteomics. The technology enables rapid identification of large numbers of proteins in a relatively short time. Comparative quantification of identified proteins from different samples is often regarded as the next step in proteomics experiments enabling the comparison of protein expression in different proteomes. Differential labeling of samples using stable isotope incorporation or conjugation is commonly used to compare protein levels between samples but these procedures are difficult to carry out in the laboratory and for large numbers of samples. Recently, comparative quantification of label-free LC(n)-MS proteomics data has emerged as an alternative approach. In this review, we discuss different computational approaches for extracting comparative quantitative information from label-free LC(n)-MS proteomics data. The procedure for computationally recovering the quantitative information is described. Furthermore, statistical tests used to evaluate the relevance of results will also be discussed.     

Proteomics of blood and derived products: what’s next?

Proteomics has changed the way proteins are analyzed in living systems. This approach has been applied to blood products and protein profiling has evolved in parallel with the development of techniques. The identification of proteins belonging to red blood cell, platelets or plasma was achieved at the end of the last century. Then, the questions on the applications emerged. Hence, several studies have focused on problems related to blood banking and products, such as the aging of blood products, identification of biomarkers, related diseases and the protein–protein interactions. More recently, a mass spectrometry-based proteomics approach to quality control has been applied in order to offer solutions and improve the quality of blood products. The current challenge we face is developing a closer relationship between transfusion medicine and proteomics. In this article, these issues will be approached by focusing first on the proteome identification of blood products and then on the applications and future developments within the field of proteomics and blood products.     

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.     

Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach

Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins.     

Proteomics of blood and derived products: what's next?

Proteomics has changed the way proteins are analyzed in living systems. This approach has been applied to blood products and protein profiling has evolved in parallel with the development of techniques. The identification of proteins belonging to red blood cell, platelets or plasma was achieved at the end of the last century. Then, the questions on the applications emerged. Hence, several studies have focused on problems related to blood banking and products, such as the aging of blood products, identification of biomarkers, related diseases and the protein-protein interactions. More recently, a mass spectrometry-based proteomics approach to quality control has been applied in order to offer solutions and improve the quality of blood products. The current challenge we face is developing a closer relationship between transfusion medicine and proteomics. In this article, these issues will be approached by focusing first on the proteome identification of blood products and then on the applications and future developments within the field of proteomics and blood products.     

Vectorial proteomics

Vectorial proteomics is a methodology for the differential identification and characterization of proteins and their domains exposed to the opposite sides of biological membranes. Proteomics of membrane vesicles from defined isolated membranes automatically determine cellular localization of the identified proteins and reduce complexity of protein characterizations. The enzymatic shaving of naturally-oriented, or specifically-inverted sealed membrane vesicles, release the surface-exposed peptides from membrane proteins. These soluble peptides are amenable to various chromatographic separations and to sequencing by mass spectrometry, which provides information on the topology of membrane proteins and on their posttranslational modifications. The membrane shaving techniques have made a breakthrough in the identification of in vivo protein phosphorylation sites in membrane proteins form plant photosynthetic and plasma membranes, and from caveolae membrane vesicles of human fat cells. This approach has also allowed investigation of dynamics for in vivo protein phosphorylation in membranes from cells exposed to different conditions. Vectorial proteomics of membrane vesicles with retained peripheral proteins identify extrinsic proteins associated with distinct membrane surfaces, as well as a variety of posttranslational modifications in these proteins. The rapid integration of versatile vectorial proteomics techniques in the functional characterization of biological membranes is anticipated to bring significant insights in cell biology.     

Global approaches to protein-protein interactions

The availability of complete, annotated genome sequences for a variety of eukaryotic organisms has paved the way for a paradigm shift in biomedical research from the 'one gene-one hypothesis' approach to more global, systematic strategies that analyse genes or proteins on a genome- and proteome-wide scale. One daunting task in the post-genome era is to determine how the complement of expressed cellular proteins - the proteome - is organised into functional, higher-order networks, by mapping all constitutive and dynamic protein-protein interactions. Traditionally, reductionist approaches have typically focused on a few, selected gene products and their interactions in a particular physiological context. In contrast, more holistic strategies aim at understanding complex biological systems, for example global protein-protein interaction networks on a cellular or organismal level. Several large-scale proteomics technologies have been developed to generate comprehensive, cellular protein-protein interaction maps.     

Environmental proteomics and metallomics

Monitoring environmental pollution using biomarkers requires detailed knowledge about the markers, and many only allow a partial assessment of pollution. New proteomic methods (environmental proteomics) can identify proteins that, after validation, might be useful as alternative biomarkers, although this approach also has its limitations, derived mainly from their application to non-model organisms. Initial studies using environmental proteomics were carried out in animals exposed to model pollutants, and led to the concept of protein expression signatures. Experiments have been carried out in model organisms (yeast, Arabidopsis, rat cells, or mice) exposed to model contaminants. Over the last few years, proteomics has been applied to organisms from ecosystems with different pollution levels, forming the basis of an environmental branch in proteomics. Another focus is connected with the presence of metals bound to biomolecules, which adds an additional dimension to metal-biomolecule and metalloprotein characterization - the field of metallomics. The metallomic approach considers the metallome: a whole individual metal or metalloid species within a cell or tissue. A metallomic analytical approach (MAA) is proposed as a new tool to study and identify metalloproteins.     

Proteomics approach and techniques in identification of reliable biomarkers for diseases

Biomarkers, also called biological markers, are indicators to identify a biological case or situation as well as detecting any presence of biological activities and processes. Proteins are considered as a type of biomarkers based on their characteristics. Therefore, proteomics approach is one of the most promising approaches in this field. The purpose of this review is to summarize the use of proteomics approach and techniques to identify proteins as biomarkers for different diseases. This review was obtained by searching in a computerized database. So, different researches and studies that used proteomics approach to identify different biomarkers for different diseases were reviewed. Also, techniques of proteomics that are used to identify proteins as biomarkers were collected. Techniques and methods of proteomics approach are used for the identification of proteins' activities and presence as biomarkers for different types of diseases from different types of samples. There are three essential steps of this approach including: extraction and separation of proteins, identification of proteins, and verification of proteins. Finally, clinical trials for new discovered biomarker or undefined biomarker would be on.     

The Plasma Proteome: High Abundance versus Low Abundance

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.     

Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.     

Mass spectrometry-based quantitative proteomics

A major aim of present-day proteomics is to study changes in protein expression levels at a global level, ideally monitoring all proteins present in cells or tissue. Mass spectrometry is a well-respected technology in proteomics that is widely used for the identification of proteins. More recently, methodologies have been introduced showing that mass spectrometry can also be used for protein quantification. This article reviews various mass spectrometry-based technologies in quantitative proteomics, highlighting several interesting applications in areas ranging from cell biology to clinical applications.     

Mass spectrometry-based quantitative proteomics

A major aim of present-day proteomics is to study changes in protein expression levels at a global level, ideally monitoring all proteins present in cells or tissue. Mass spectrometry is a well-respected technology in proteomics that is widely used for the identification of proteins. More recently, methodologies have been introduced showing that mass spectrometry can also be used for protein quantification. This article reviews various mass spectrometry-based technologies in quantitative proteomics, highlighting several interesting applications in areas ranging from cell biology to clinical applications.     

Comprehensive analysis of the mouse renal cortex using two-dimensional HPLC – tandem mass spectrometry

Background  

Proteomic methodologies increasingly have been applied to the kidney to map the renal cortical proteome and to identify global changes in renal proteins induced by diseases such as diabetes. While progress has been made in establishing a renal cortical proteome using 1-D or 2-DE and mass spectrometry, the number of proteins definitively identified by mass spectrometry has remained surprisingly small. Low coverage of the renal cortical proteome as well as our interest in diabetes-induced changes in proteins found in the renal cortex prompted us to perform an in-depth proteomic analysis of mouse renal cortical tissue.     

Proteomics: current techniques and potential applications to lung disease

Proteomics aims to study the whole protein content of a biological sample in one set of experiments. Such an approach has the potential value to acquire an understanding of the complex responses of an organism to a stimulus. The large vascular and air space surface area of the lung expose it to a multitude of stimuli that can trigger a variety of responses by many different cell types. This complexity makes the lung a promising, but also challenging, target for proteomics. Important steps made in the last decade have increased the potential value of the results of proteomics studies for the clinical scientist. Advances in protein separation and staining techniques have improved protein identification to include the least abundant proteins. The evolution in mass spectrometry has led to the identification of a large part of the proteins of interest rather than just describing changes in patterns of protein spots. Protein profiling techniques allow the rapid comparison of complex samples and the direct investigation of tissue specimens. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. These methodologies have made the application of proteomics on the study of specific diseases or biological processes under clinically relevant conditions possible. The quantity of data that is acquired with these new techniques places new challenges on data processing and analysis. This article provides a brief review of the most promising proteomics methods and some of their applications to pulmonary research.