Cancer diagnosis using proteomic patterns

The advent of proteomics has brought with it the hope of discovering novel biomarkers that can be used to diagnose diseases, predict susceptibility and monitor progression. Much of this effort has focused upon the mass spectral identification of the thousands of proteins that populate complex biosystems such as serum and tissues. A revolutionary approach in proteomic pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian cancer. Proteomic pattern analysis relies on the pattern of proteins observed and does not rely on the identification of a traceable biomarker. Hundreds of clinical samples per day can be analyzed utilizing this technology, which has the potential to be a novel, highly sensitive diagnostic tool for the early detection of cancer.     

High-content affinity-based proteomics: unlocking protein biomarker discovery

Single protein biomarkers measured with antibody-based affinity assays are the basis of molecular diagnostics in clinical practice today. There is great hope in discovering new protein biomarkers and combinations of protein biomarkers for advancing medicine through monitoring health, diagnosing disease, guiding treatment, and developing new therapeutics. The goal of high-content proteomics is to unlock protein biomarker discovery by measuring many (thousands) or all (～23,000) proteins in the human proteome in an unbiased, data-driven approach. High-content proteomics has proven technically difficult due to the diversity of proteins, the complexity of relevant biological samples, such as blood and tissue, and large concentration ranges (in the order of 10(12) in blood). Mass spectrometry and affinity methods based on antibodies have dominated approaches to high-content proteomics. For technical reasons, neither has achieved adequate simultaneous performance and high-content. Here we review antibody-based protein measurement, multiplexed antibody-based protein measurement, and limitations of antibodies for high-content proteomics due to their inherent cross-reactivity. Finally, we review a new affinity-based proteomic technology developed from the ground up to solve the problem of high content with high sensitivity and specificity. Based on a new generation of slow off-rate modified aptamers (SOMAmers), this technology is unlocking biomarker discovery.     

Proteomic patterns as a diagnostic tool for early-stage cancer: a review of its progress to a clinically relevant tool.

The pace of development in novel technologies that promise improvements in the early diagnosis of disease is truly impressive. One such technology at the forefront of this revolution is mass spectrometry. New capabilities in mass spectrometry have provided the means for the development of proteomics, and the race is on to find innovative ways to apply this powerful technology to solving the problems faced in clinical medicine. One area that has garnered much attention over the past few years is the use of mass spectral patterns for cancer diagnostics. The use of these so-called 'proteomic patterns' for disease diagnosis relies fundamentally on the pattern of signals observed within a mass spectrum rather than the more conventional identification and quantitation of a biomarker such as in the case of cancer antigen-125- or prostate-specific antigen. The inherent throughput of proteomic pattern technology enables the analysis of hundreds of clinical samples per day. Currently, there are two primary means by which proteomic patterns can be acquired, surface-enhanced laser desorption/ionization (SELDI) and an electrospray ionization (ESI) method that has been popularized under the name, OvaCheck. In this review, an historical perspective on the development of proteomic patterns for the diagnosis of early-stage cancers is described. In addition, a critical assessment of the overall technology is presented with an emphasis on the steps required to enable proteomic pattern analysis to become a viable clinical tool for diagnosing early-stage cancers.     

Urinary proteomics

Protein measurement in urine has been used for many years for the diagnosis and monitoring of renal disease. The pattern of urinary protein excretion can be used to identify the cause of the disease and to classify proteinuria. In recent years, proteomics has proven to be a powerful tool in investigation and clinical medicine. Proteomics employs a protein separation method and the identification of proteins using mass spectrometry. One of the objectives of clinical proteomics is the identification of biological markers of disease. To accomplish this, it is necessary to have a normal proteome of the medium in question, which in our case is urine. Comparison of the normal urinary proteome with the urinary proteome from patients with a defined disease can detect proteins expressed differentially from one another. The aim of this review is to present the situation of urinary proteomics, putting special emphasis on its application in the diagnosis of glomerular diseases, renal allograft rejection, urological cancers and urolithiasis.     

The clinical application of proteomics

BACKGROUND: Proteomics is defined as a scientific approach used to elucidate all protein species within a cell or tissue, and many researchers are taking advantage of proteomic technology to elucidate protein changes between healthy and diseased states. METHODS: The application of proteomic techniques and strategies to the field of medicine is slowly transforming the way biomarker discovery is conducted. However, the complexity of serum is the source of both its promise to clinical applications and its challenge to proteomic analysis. Like any new technology when it is first introduced, proteomics has been touted with much hope and promise. RESULTS AND CONCLUSIONS: We provide a review of the clinical application of proteomics with the emphasis on current practical issues and challenges facing proteomic research.     

Mass spectrometry-based membrane proteomics in cancer biomarker discovery

Membrane proteins are involved in central processes such as cell signaling, cell–cell interactions and communication, ion and metabolite transport and in general play a crucial role in cell homeostasis. Cancer and cancer metastasis have been correlated to protein expression levels and dysfunction, with membrane proteins playing an important role, and are thus used as drug targets and potential biomarkers for prognostic or diagnostic purposes. Despite the critical biological significance of membrane proteins, proteomic analysis has been a challenging task due to their hydrophobicity. In this review, recent advances in the proteomic analysis of membrane proteins are presented, focusing on membrane fraction enrichment techniques combined with labeled or label-free shotgun proteomics approaches for the identification of potential cancer biomarkers.     

Proteomics: a new diagnostic frontier

BACKGROUND: Analysis of proteins has been an integral part of the field of clinical chemistry for decades. Recent advances in technology and complete identification of the human genome sequence have opened up new opportunities for analysis of proteins for clinical diagnostic purposes. METHODS: Content of a recent conference of proteomics is summarized. RESULTS: New analytical methods allow the simultaneous analysis of a large number of proteins in biological fluids such as serum and plasma, offering partial views of the complete set of proteins or proteome. Plasma presents many analytical challenges, such as the complexity of components, predominance of a few major components, and the large concentration range of components, but the number of proteins that can be detected in plasma has expanded dramatically from hundreds to thousands. At the same time, there is increased capability to detect structural variations of proteins. Recent studies also identified the presence of complex sets of small protein fragments in plasma. This set of protein fragments, the fragmentome or peptidome, is potentially a rich source of information about physiologic and disease processes. CONCLUSIONS: Advances in proteomics offer great promise for the discovery of markers that might serve as the basis for new clinical laboratory tests. There are many challenges, however, in the translation of newly discovered markers into clinical laboratory tests.     

Proteomics: the next revolution in laboratory medicine?

BACKGROUND: The identification of specific genetic alterations and protein profiles associated with disease offers a unique opportunity to develop proteomics-based assays for early diagnosis. By identifying proteins in serum/plasma, a minimally invasive tool is used to assess the presence of disease and to monitor response to treatment and/or disease progression. The potential clinical applications of this tool are broad-based, including the diagnosis not only of cancer but also cardiovascular and neuromuscular diseases, organ transplantation associated conditions, and infertility. METHODS: A number of competing chromatographic techniques have been proposed for overcoming the complexity and labor-intensive manipulations associated with the traditional technique for proteomic analysis, which is based on two-dimensional gel electrophoretic techniques. However, mass spectrometry has now assumed a central role in most proteomic workflows, and several combinations of ionization sources, analyzers and fragmentations devices have been described and developed. RESULTS: Thanks to proteomic applications in the diagnosis of cancer, several research groups have identified proteomic patterns associated with ovarian, prostatic, colorectal and other cancers. While the sensitivity and specificity of these patterns are highly satisfactory, there are still some open questions concerning the standardization, reproducibility, and inter-laboratory agreement of these data. CONCLUSIONS: Proteomics, and, in particular, serum mass spectroscopic proteomic pattern diagnostics, is a rapid expanding field of research. The plasma proteoma has an important position at the intersection between genes and diseases, and clinical laboratories must adapt to a new era of tests based on proteomics and genomics. In the future, mass spectrometry will become an essential tool in the clinical laboratory.     

Label-free mass spectrometry-based proteomics for biomarker discovery and validation

For most diseases, better biomarkers are urgently needed to enable (early) detection, diagnosis, prognosis, stratification for therapy and response monitoring. Proteomics delineates gene products that carry out the majority of cellular functions, and thereby may not only yield insight into altered signaling pathways in disease, but also yield novel biomarkers. In recent years, great progress has been made in mass spectrometry-based analysis of clinical tissues and biofluids, with identification and quantification of thousands of proteins now becoming increasingly routine. However, biomarker validation and clinical translation has turned out to be challenging. In this review, we summarize current mass spectrometry-based proteomics strategies for biomarker discovery and verification using selected reaction monitoring, with a focus on progress and recent applications in clinical material using label-free approaches.     

Vascular proteomic mapping in vivo

Summary.  Molecular targeting of drugs and imaging agents remain important yet elusive goals in modern medicine. Technological advancements in genomics and proteomics methods have detected differentially expressed genes and proteins, uncovering many new candidate targets in a wide array of diseases and tissues. However, methods to validate potential targets in vivo tend to be quite laborious so that the validation and testing phase has become rate-limiting in bringing treatments to the clinic. There is a critical need for integrated approaches combining state-of-the-art methodologies in proteomics and in vivo imaging to accelerate validation of newly discovered vascular targets for nanomedicines, drugs, imaging agents, and gene vectors. This paper is a review of vascular targeting and proteomics, and will present recent developments in proteomic imaging. A new in vivo organellar proteomic imaging platform will be discussed, which combines subcellular fractionation, mass spectrometry, bioinformatic database interrogation, monoclonal antibody technology and a battery of imaging modalities to rapidly discover and validate tissue-specific endothelial protein targets in vivo . Technological advancements are permitting large-scale proteomic mapping to be performed. New targets have been discovered that permit organ-specific targeting in vivo . Improvements in imaging are creating standards for validation of targets in vivo . Tumor imaging and radioimmunotherapy have also been improved through these efforts. Although we are moving towards a comprehensive mapping of the protein expression by the endothelium, much more needs to be done.     

Genomics and proteomics approaches in understanding tumor angiogenesis

Functional genomic and proteomic approaches have begun to revolutionize cancer research. The advent of powerful technologies, such as DNA microarrays, serial analysis of gene expression, RNA interference and proteomics, has accelerated investigations of gene identification and function at a scale never before accomplished. Approaches integrating these technologies with high-throughput forward and reverse genetic screens, are already providing insights into the mechanistic understanding of angiogenesis, leading to the identification of proteins that can be used for selective targeting of tumor vessels.     

Direct-tissue SELDI-TOF mass spectrometry analysis: a new application for clinical proteomics

BACKGROUND: New molecular profiling technologies can aid in analysis of small pathologic samples obtained by minimally invasive biopsy and may enable the discovery of key biomarkers synergistic with anatomopathologic analysis related to prognosis, therapeutic response, and innovative target validation. Thus proteomic analysis at the histologic level in healthy and pathologic settings is a major issue in the field of clinical proteomics. METHODS: We used surface-enhanced laser desorption ionization-time-of-flight mass spectrometry (SELDI-TOF MS) technology with surface chromatographic subproteome enrichment and preservation of the spatial distribution of proteomic patterns to detect discrete modifications of protein expression. We performed in situ proteomic profiling of mouse tissue and samples of human cancer tissue, including brain and lung cancer. RESULTS: This approach permitted the discrimination of glioblastomas from oligodendrogliomas and led to the identification of 3 potential markers. CONCLUSION: Direct tissue proteomic analysis is an original application of SELDI-TOF MS technology that can expand the use of clinical proteomics as a complement to the anatomopathological diagnosis.     

外泌体蛋白质组学分析在心血管疾病中应用的研究进展

外泌体是由各种类型细胞从晚期内质体分泌的大小为30～100 nm的细胞外囊泡结构。多项研究显示外泌体在多种人类疾病尤其是心血管疾病中发挥着重要的作用。近来的研究显示外泌体蛋白质组学分析的方法能够成功鉴定多种外泌体相关的蛋白质,并且有助于揭示其作用效应的新机制。该文将从分析评估不同蛋白质组学技术在鉴定外泌体蛋白中的应用并讨论蛋白组学分析外泌体在心血管疾病研究中的最新进展进行综述。     

Platelet proteomics

Summary.  As anucleate cell particles, platelets do not lend themselves to analysis by traditional cell and molecular biology techniques. Moreover, while valuable information may be gleaned from studies of messenger RNA in platelets, the rapid events in platelets are not governed by or dependent on alterations in gene expression. In contrast, proteomics, the study of the protein complement of a genome, will have a major impact on platelet biology. It offers the opportunity to comprehensively describe the proteins involved in discrete elements of platelet function, from the subsecond events following platelet activation and adhesion through to platelet aggregation and granule secretion. As the function of every protein is understood and as the mechanisms that regulate protein modifications are unravelled, we will discover a wealth of proteins that are themselves potential therapeutic agents or novel targets for the development of diagnostics and drugs. Here we review the current applications of proteomics to platelet research. We briefly describe various proteomic approaches to unravel platelet biology, including the documentation of platelet proteins, the investigation of thrombin-activated phosphotyrosine signaling networks and the analysis of the proteins that are secreted upon platelet activation. Proteomics is a young field and there are only a handful of published examples applying proteomics to platelet research. This number will increase over the next few years, as advances in analytical methods allow a more functional analysis of the platelet proteome.     

The use of proteomics for the assessment of clinical samples in research

Proteomics, the analysis of expressed proteins, has been an important developing area of research for the past two decades [Anderson, NG, Anderson, NL. Twenty years of two-dimensional electrophoresis: past, present and future. Electrophoresis 1996;17:443-453]. Advances in technology have led to a rapid increase in applications to a wide range of samples; from initial experiments using cell lines, more complex tissues and biological fluids are now being assessed to establish changes in protein expression. A primary aim of clinical proteomics is the identification of biomarkers for diagnosis and therapeutic intervention of disease, by comparing the proteomic profiles of control and disease, and differing physiological states. This expansion into clinical samples has not been without difficulties owing to the complexity and dynamic range in plasma and human tissues including tissue biopsies. The most widely used techniques for analysis of clinical samples are surface-enhanced laser desorption/ionisation mass spectrometry (SELDI-MS) and 2-dimensional gel electrophoresis (2-DE) coupled to matrix-assisted laser desorption ionisation [Person, MD, Monks, TJ, Lau, SS. An integrated approach to identifying chemically induced posttranslational modifications using comparative MALDI-MS and targeted HPLC-ESI-MS/MS. Chem. Res. Toxicol. 2003;16:598-608]-mass spectroscopy (MALDI-MS). This review aims to summarise the findings of studies that have used proteomic research methods to analyse samples from clinical studies and to assess the impact that proteomic techniques have had in assessing clinical samples.     

Oncoproteomics

Researchers have long acknowledged that changes in genes or gene activity lead to cancer. However, it was difficult to understand the function of such specific genes and their interaction in communication networks and the roles played by their protein products in molecular pathways. Protein molecules have direct influences on the development of cancer as it fundamentally arises due to aberrant signaling pathways. Identifying and understanding these changes is the primary theme of cancer proteomics, also termed as oncoproteomics. The ultimate objective of oncoproteomics is to acclimatize proteomic technologies for regular use in clinical laboratories for the purpose of diagnostic and prognostic categorization of disease condition, as well as in assessing drug toxicity and efficiency. Information gained from such technologies may soon exert a spectacular change in cancer research and impact dramatically on the care of cancer patients. Investigations of tumor-specific proteomic profiles may also allow better understanding of tumor development and the identification of novel targets for cancer therapy. In this review, we have tried to offer a wide perspective on recent progresses in proteomic research strategies, their applications in the discovery of novel tumor markers and drug targets and their role in illustrating action mechanisms of biomarkers and anticancer drugs including drug resistance.     

FTICR mass spectrometry in proteomics

Analytical tools that allow rapid screening, low sample consumption and accurate protein identification are of great importance in studies of complex biological samples. Today, mass spectrometry (MS) is a key analytical tools with applications in a wide variety of fields, reaching from the analysis of elemental compositions in various materials to the identification of large protein complexes. One of the fastest growing fields of MS applications is proteomics, or the study of protein expression in an organism. In the traditional proteomic approach, two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis is applied for the separation and visualization of proteins. In this review, the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry, including up-front multidimensional liquid separations for 'top down' or 'bottom up' proteomic approaches, are presented.     

应用蛋白质芯片技术筛选直肠癌患者

目的应用表面增强激光解吸电离飞行时间质谱技术(SELDI-TOF-MS)从直肠癌患者血清中筛选标志蛋白,找出最佳的标志蛋白组合模式作为临床诊断指标。方法采用WCX2芯片及SELDI-TOF-MS技术对98例直肠癌患者及40例对照组血清进行蛋白质指纹图谱检测分析,所得到的结果采用Biomarker Wizard和Biomarker Patterns System软件分析。结果直肠癌组与对照组共有26个蛋白质差异有显著性(P<0.05);以其中4个蛋白质生物标志物(质/荷比9295、3730、3938和4095)组建筛选模型,经盲法验证,其敏感度为95.0%(57/60),特异性为93.4%(45/48);CEA敏感度为68.3%(41/60),特异性为77.1%(37/48);CA199敏感度为58.3%(35/60),特异性为75%(36/48)。结论SELDI-TOF-MS技术的敏感度和特异性远远高于目前所采用的某一单独的标志物的血清学诊断,其结果对进一步研究直肠癌的蛋白质组学改变及其临床应用可能具有重要意义。     

Discovery of disease-induced post-translational modifications in cardiac contractile proteins

Post-translational modifications to myofilament proteins are essential for the regulation of cardiac function in both normal and disease states. Recent developments in the field of proteomics have produced a variety of useful tools to study protein modifications. Current applications of proteomic technologies in the study of modifications to myofilament proteins are summarized. The separation, identification and characterization of myofilament modifications using gel electrophoresis and mass spectrometry approaches are discussed. Each method is illustrated and evaluated with selected examples, and several powerful emerging technologies are assessed.     

Bioinformatics strategies for proteomic profiling

Clinical proteomics is an emerging field that involves the analysis of protein expression profiles of clinical samples for de novo discovery of disease-associated biomarkers and for gaining insight into the biology of disease processes. Mass spectrometry represents an important set of technologies for protein expression measurement. Among them, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF-MS), because of its high throughput and on-chip sample processing capability, has become a popular tool for clinical proteomics. Bioinformatics plays a critical role in the analysis of SELDI data, and therefore, it is important to understand the issues associated with the analysis of clinical proteomic data. In this review, we discuss such issues and the bioinformatics strategies used for proteomic profiling.