Pathway Proteomics

Biological pathways represent the relationships (reactions and interactions) between biological molecules in the context of normal cellular functions and disease mechanisms. Understanding the roles of proteins and signaling pathways expressed within disease, and their link to drug discovery and drug development are central in today's target-driven pharmaceutical processes. This article gives an overview of proteomics strategies, including global expression analysis as well as focused approaches using multidimensional separation by both gel- and liquid-phase techniques linked to mass spectrometry, as applied to two of the pathways involved in inflammatory diseases. In primary human cell studies, our group has annotated and identified thousands of proteins using both electrospray ionization and matrix-assisted laser desorption ionization (MALDI)-sequencing technology. Annotations made from gel images and chromatography fractionation, interfaced to high-end mass spectrometry sequence and structure identity, are cornerstones in cutting-edge protein expression profiling. Regarding phosphorylation mechanisms of kinases, the quantitative stoichiometry can be determined using affinity probe isolations. Another strategy involves micro-preparative sample processing, which has been used to analyze single-target phosphoproteins and their relative phospho-stoichiometry.     

The use of proteomics for systematic analysis of normal and transformed hematopoietic stem cells

Blood cell production involves the commitment and differentiation of hematopoietic stem cells to committed progenitor cells which undergo a programmed development to form mature cells such as neutrophils, macrophages and lymphocytes. This complex process can be disrupted in diseases such as the leukemias and myeloproliferative disorders by oncogenes such as protein tyrosine kinases. The analysis of expression patterns for specific genes suggests that the regulation of protein expression can be achieved in a posttranslational fashion. Post-translational protein modification, such as phosphorylation and acetylation govern events in blood cell production and yet cannot be measured using conventional molecular biology approaches. For this reason a suite of techniques in mass spectrometry needs to be applied to define regulation and disregulation in normal and abnormal hematopoiesis. These approaches include discovery proteomics with relative quantification of thousands of proteins. Alternatively targeted examination of a single protein to identify its interaction partners or post-translational modifications using mass spectrometry reveals much mechanistic detail. The use of mass spectrometry and proteomics approaches in stem cell and leukemia studies has thus far revealed a good deal of information on hematopoiesis. Further application of the proteomics approach is a necessity to gain true insight into regulatory processes governing the production of billions of blood cells a day, and ways in which that process can be manipulated to therapeutic advantage.     

Methods for investigation of targeted kinase inhibitor therapy using chemical proteomics and phosphorylation profiling

Phosphorylation acts as a molecular switch for many regulatory events in signaling pathways that drive cell division, proliferation, and apoptosis. Because of the critical nature of these protein post-translational modifications in cancer, drug development programs often focus on inhibitors for kinases and phosphatases, which control protein phosphorylation. Numerous kinase inhibitors have entered clinical use, but prediction of their efficacy and a molecular basis for patient response remain uncertain. Chemical proteomics, the combination of drug affinity chromatography with mass spectrometry, identifies potential target proteins that bind to the drugs. Phosphorylation profiling can complement chemical proteomics by cataloging modifications in the target kinases and their downstream substrates using phosphopeptide enrichment and quantitative mass spectrometry. These experiments shed light on the mechanism of disease development and illuminate candidate biomarkers to guide personalized therapeutic strategies. In this review, commonly applied technologies and workflows are discussed to illustrate the role of proteomics in examining tumor biology and therapeutic intervention using kinase inhibitors.     

蛋白质组学技术在药物肝毒性研究中的应用

蛋白质组学技术的发展促进了许多学科的发展,毒理蛋白质组学(toxicoproteomics)整合了经典毒理学、病理学和蛋白质差异表达分析技术,使毒理学研究上升到了一个新的高度。药物性肝损伤是肝脏疾病的一个重要原因,也是药物开发所面临的一项重大挑战。现就蛋白质组学的主要技术,包括双向凝胶电泳-质谱(2-DE-MS)、液相色谱-串联质谱(LC-MS-MS)、保留色谱-质谱(RC-MS)和蛋白质阵列,以及其在药物肝毒性研究中的应用,包括毒性蛋白标志物的筛选、毒性机制研究、毒性预测与毒性蛋白质数据库的建立方面进行综述。     

Mass spectrometry in toxinology: a 21st-century technology for the study of biopolymers from venoms.

Mass spectrometry, developed in the early days of the 20th century for the structural analysis of ions from organic compounds, has evolved from an analytical technique almost entirely applied to structural studies of small molecules, to a diversified technology that is now increasingly focused on the study of biological macromolecules. Novel instrument developments and appropriate ionization techniques have permitted the application of mass spectrometry to the analysis of biopolymers such as proteins, sugars and nucleic acids and have opened the door to a multiplicity of applications, and not the least being proteomics. Increasingly used as a basic analytical tool in biology laboratories, mass spectrometry has now found another niche of application in the field of venom and toxin studies. The technique is well suited to the analysis of peptide and protein components of venoms, be it for global mass mapping of complex mixtures or structural studies on individual toxins. Further enhanced by hyphenation with separation technologies, mass spectrometry is well adapted to de-convolve the extreme complexity of natural venoms and biological extracts in which toxinologists specialize. This special issue highlights a number of applications of mass spectrometry in this field and presents some of the most recent work illustrating the benefits of various state-of-the-art mass spectrometry technologies for the study of animal venoms and toxins.     

Functional proteomics to study protection of the ischaemic myocardium

Mechanisms to reduce the deleterious effects of myocardial ischaemia are of particular clinical importance and have been the focus of intense research for a number of years. Among novel approaches to studying the ischaemic heart, proteomics, or the analysis of all cellular proteins, presents as a powerful method to deconstruct the mechanisms of disease and protection. Specifically, the field of functional proteomics is an emerging application of proteomics that melds aspects of classical proteomics, biochemistry, molecular biology and physiology into an approach that facilitates an understanding of how proteins and protein interactions engender phenotype. This review highlights different types of proteomic applications and provides a prospectus for functional proteomics as a robust vehicle driving drug discovery and design.     

Functional proteomics to study protection of the ischaemic myocardium

Mechanisms to reduce the deleterious effects of myocardial ischaemia are of particular clinical importance and have been the focus of intense research for a number of years. Among novel approaches to studying the ischaemic heart, proteomics, or the analysis of all cellular proteins, presents as a powerful method to deconstruct the mechanisms of disease and protection. Specifically, the field of functional proteomics is an emerging application of proteomics that melds aspects of classical proteomics, biochemistry, molecular biology and physiology into an approach that facilitates an understanding of how proteins and protein interactions engender phenotype. This review highlights different types of proteomic applications and provides a prospectus for functional proteomics as a robust vehicle driving drug discovery and design.     

Reaching for the deep proteome: Recent nano liquid chromatography coupled with tandem mass spectrometry-based studies on the deep proteome

In the last decade, there has been a dramatic progress in separation techniques, mass spectrometry, and bioinformatics, and this progress has significantly improved the techniques on protein analysis. However, the analysis of low-abundance proteins is still challenging because of the limited performance in the method of choice compared to the complexity and the vast dynamic range of biological samples. Since this issue is a big obstacle in most proteomics investigations, great interest has been paid recently to various techniques, such as multi-dimensional analysis, specific peptide selection, high-abundance protein depletion, ligand library treatment, to address this challenge. Therefore, here, the author reviews recent nano liquid chromatography coupled with tandem mass spectrometry-based studies on the deep proteome, mainly focusing on their methods and perspectives.     

Pharmacogenomics of cardiovascular pharmacology: molecular network analysis in pleiotropic effects of statin -- an experimental elucidation of the pharmacologic action from protein-protein interaction analysis

The ebb and flow of cellular life depends largely on signaling pathways and networks, which are regulated by specific protein-protein interactions. These interactions often involve assembly of large signaling complexes containing many different protein kinases, protein phosphatases, their substrates, and scaffold proteins. Identification of protein complexes is the key to understanding cellular functions. One of the techniques used for the isolation of protein complexes is the affinity purification system. Inhibitors of 3-hydroxyl-3-methyglutaryl coenzyme A (HMG-CoA) reductase (i.e., statins) exert cholesterol-independent vasoprotective effects that are mediated, in part, through the activation of Akt. However, the molecular mechanism remains unknown. To elucidate the molecular mechanisms of the pleioptropic effects of statins, we searched for the binding molecule of Akt1 by using a combined mass spectrometry and affinity purification strategy. By this technique, we identified the protein-protein interactions of 23 proteins from statin-treated rat aortic endothelial cells (rAECs). Our results suggest that this approach is very effective and statin activates many Akt down-stream targets, not only endothelial nitric oxide synthase (eNOS). The methodology presented here would provide a new tool for chemical proteomics in medicinal science.     

Mass Spectrometry-Based Targeted Proteomics as a Tool to Elucidate the Expression and Function of Intestinal Drug Transporters

Intestinal transporter proteins affect the oral bioavailability of many drugs in a significant manner. In order to estimate or predict their impact on oral drug absorption, data on their intestinal expression levels are needed. So far, predominantly mRNA expression data are available which are not necessarily correlated with the respective protein content. All available protein data were assessed by immunoblotting techniques such as Western blotting which both possess a number of limitations for reliable protein quantification. In contrast to this, mass spectrometry-based targeted proteomics may represent a promising alternative method to provide comprehensive protein expression data. In this review, we will summarize so far available intestinal mRNA and protein expression data for relevant human multidrug transporters. Moreover, recently observed mass spectrometry-based targeted proteomic data will be presented and discussed with respect to potential functional consequences. Associated to this, we will provide a short tutorial how to set up these methods and emphasize critical aspects in method development. Finally, potential limitations and pitfalls of this emerging technique will be discussed. From our perspective, LC-MS/MS-based targeted proteomics represents a valuable new method to comprehensively analyse the intestinal expression of transporter proteins. The resulting expression data are expected to improve our understanding about the intestinal processing of drugs.     

Capillary electrophoresis–mass spectrometry: Recent trends in clinical proteomics

The increasing attention now paid to the elucidation of human proteome strengthened the development of analytical instruments able to provide reliable proteins and peptides quantitation and characterization in biological fluids and tissues. Emerging from proteomics, clinical proteomics exclusively considers its biomedical applications. It evaluates, often by high-throughput comparative platforms, the protein and peptide variations in body fluids, cells and tissues under different physiological and pathological conditions with the aim of discovering disease biomarkers. Among the available analytical methodologies, mass spectrometry in coupling with liquid chromatography or capillary electrophoresis demonstrated to be the eligible technique for protein detection and identification. This review summarizes the most recent applications of capillary electrophoresis–mass spectrometry to clinical proteomics, focusing on capillary zone electrophoresis separation mode and ESI and MALDI ionizations, which are the most frequently applied capillary electrophoresis–mass spectrometry hyphenated techniques.     

Quantitative and targeted proteomics-based identification and validation of drug efficacy biomarkers

Proteomics refers to the large-scale study of proteins, providing comprehensive and quantitative information on proteins in tissue, blood, and cell samples. In many studies, proteomics utilizes liquid chromatography-mass spectrometry. Proteomics has developed from a qualitative methodology of protein identification to a quantitative methodology for comparing protein expression, and it is currently classified into two distinct methodologies: quantitative and targeted proteomics. Quantitative proteomics comprehensively identifies proteins in samples, providing quantitative information on large-scale comparative profiles of protein expression. Targeted proteomics simultaneously quantifies only target proteins with high sensitivity and specificity. Therefore, in biomarker research, quantitative proteomics is used for the identification of biomarker candidates, and targeted proteomics is used for the validation of biomarkers. Understanding the specific characteristics of each method is important for conducting appropriate proteomics studies. In this review, we introduced the different characteristics and applications of quantitative and targeted proteomics, and then discussed the results of our recent proteomics studies that focused on the identification and validation of biomarkers of drug efficacy. These findings may enable us to predict the outcomes of cancer therapy and drug-drug interactions with antibiotics through changes in the intestinal microbiome.     

Application of MALDI-TOF mass spectrometry in screening and diagnostic research

During the last years, mass spectrometry has revolutionised protein biochemistry and has advanced to a superior tool for the identification and detailed analysis of peptides and proteins. The high throughput allowed by some mass spectrometry platforms has enabled the important step from analysis of individual proteins to proteomics. Recently, an additional field of mass spectrometry applications has emerged - namely screening and diagnostic research. In contrast to protein identification, screening applications have to detect analyte molecules of defined molecular weights which can be calculated beforehand, for example by means of chemical structures. Here, the accuracy and sensitivity of mass spectrometry has to be combined with the requirements of high-throughput analyses, in particular speed and automation. These criteria are especially fulfilled by state of the art matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) instruments. The first high throughput screening (HTS) application proved to be genotyping of single nucleotide polymorphisms. The same principle was later applied for several quality control issues, for example for oligonucleotides, peptide or compound libraries. This development has culminated in the screening and profiling of complex biomarker patterns in clinical proteomics to detect a molecular fingerprint for specific diseases in biological samples. Thus, mass spectrometry based methods are expected to enable a very early diagnosis of diseases with minimally invasive methods of investigation. This type of high end screening application has the potential to revolutionise the early diagnosis of many diseases. Here, we give an overview of the application of mass spectrometry in the fields of screening and diagnostic research.     

Separation of biological proteins by liquid chromatography

After the success of human genome project, proteome is a new emerging field of biochemistry as it provides the knowledge of enzymes (proteins) interactions with different body organs and medicines administrated into human body. Therefore, the study of proteomics is very important for the development of new and effective drugs to control many lethal diseases. In proteomics study, analyses of proteome is essential and significant from the pathological point of views, i.e., in several serious diseases such as cancer, Alzheimer’s disease and aging, heart diseases and also for plant biology. The separation and identification of proteomics is a challenging job due to their complex structures and closely related physico-chemical behaviors. However, the recent advances in liquid chromatography make this job easy. Various kinds of liquid chromatography, along with different detectors and optimization strategies, have been discussed in this article. Besides, attempts have been made to include chirality concept in proteomics for understanding mechanism and medication of various disease controlled by different body proteins.Abbreviations: ACN, acetonitrile; AIEC, anion exchange chromatography; CEC, capillary electro-chromatography; CIEF, capillary isoelectric focusing; CSF, cerebrospinal fluid; 2D-nano LC, two-dimensional nano liquid chromatography quadrupole; Q-TOFMS/MS, time-of-flight tandem-mass spectrometry; EC, electro-chromatography; ESI-LC–MS, electrospray ionization liquid chromatography–mass spectrometry; FA, formic acid; FLP, FMRF amide-like peptide; GPI-APs, glycosylphosphadylinositol anchored proteins; GSH, glutathione stimulating hormone; GSTs, glutathione-S-transferase isoenzyme; HFBA, heptafluorobutyric acid; HPLC, high performance liquid chromatography; ICAT, isotope coded affinity tag; IEF-SEC, isoelectrofocussing size-exclusion chromatography; IMCD, inner medullary collecting duct; LC–MS, liquid chromatography–mass spectrometry; LC-Q-TOF, liquid chromatography-quadrupole time-of-flight tandem mass; MS/MS, spectrometry; LC-dual ESI, liquid chromatography dual electrospray ionization-Fourier transform; FT-ICR-MS, ion cyclotron resonance-mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization-time-of flight; MFGM, milk fat globule membranes; MMA, mass measurement accuracy; MPC, mesenchymal progenitor cell; NLFs, Nasal lavage fluids; NLP, neuropeptide like protein; PC2, prohormone convertase-2; PS II, photosystem II; RPLC, reversed phase liquid chromatography; SCX, strong cation exchange; SEC, size-exclusion chromatography; TFA, trifluoroacetic acid; TIC, total ion current; TRAF, tumor necrosis factor receptor     

The continuing evolution of shotgun proteomics

Shotgun proteomics has emerged as a powerful approach for the analysis of complex protein mixtures, including biofluids, tissues, cells, organelles or protein complexes. Having evolved from the integration of chromatography and mass spectrometry, innovations in sample preparation, multidimensional chromatography, mass spectrometry and proteomic informatics continually facilitate, enable and challenge shotgun proteomics. As a result, shotgun proteomics continues to evolve and enable new areas of biological research, and is beginning to impact human disease diagnosis and therapeutic intervention.     

Fractionation of peptides and identification of proteins from Saccharomyces cerevisiae in proteomics with the use of reversed-phase capillary liquid chromatography and pI-based approach

The aim of the work was to explore the identification of proteins from Saccharomyces cerevisiae using combined capillary reversed-phase liquid chromatography (RPLC) and in-solution isoelectric focusing (sIEF) for fractionation of peptides prior to mass spectrometry analysis. That method was proved to be the alternative separation method for complex mixtures of protein tryptic digests in proteomics. Analysis of the identification of peptides was performed with the use of electrospray ionization-ion trap tandem mass spectrometry (ESI-IT-MS/MS). First, the sIEF fractionation was carried out prior to separation and mass spectrometry identification by nano-LC/ESI-MS/MS instrument. The proposed approach based on sIEF and nano-LC/ESI-MS/MS analysis was proved to be an efficient and accurate alternative fractionation method of complex protein digests and can be considered as the useful tool for identification of proteins. Moreover, analytical information from that approach can be considered as the additional source of database matching constraint and can be valuable tool for analytical and bioinformatics studies of peptides fractionation in proteomics. Based on the MS/MS results obtained with ESI-IT-MS/MS instrument, 851 proteins from S. cerevisiae were identified. However, after careful analysis of the data reduction in number of proteins to 126 was obtained. Those results are discussed and interpreted in the view of the evaluation method used.     

Electrochemistry-mass spectrometry in drug metabolism and protein research

The combination of electrochemistry coupled on-line to mass spectrometry (EC-MS) forms a powerful analytical technique with unique applications in the fields of drug metabolism and proteomics. In this review the latest developments are surveyed from both instrumental and application perspectives. The limitations and solutions for coupling an electrochemical system to a mass spectrometer are discussed. The electrochemical mimicking of drug metabolism, specifically by Cytochrome P450, is high-lighted as an application with high biomedical relevance. The EC-MS analysis of proteins also has promising new applications for both proteomics research and biomarker discovery. EC-MS has furthermore advantages for improved analyte detection with mass spectrometry, both for small molecules and large biomolecules. Finally, potential future directions of development of the technique are briefly discussed.