Approaching MALDI molecular imaging for clinical proteomic research: current state and fields of application

MALDI imaging mass spectrometry (‘MALDI imaging’) is an increasingly recognized technique for biomarker research. After years of method development in the scientific community, the technique is now increasingly applied in clinical research. In this article, we discuss the use of MALDI imaging in clinical proteomics and put it in context with classical proteomics techniques. We also highlight a number of upcoming challenges for personalized medicine, development of targeted therapies and diagnostic molecular pathology where MALDI imaging could help.     

Approaching MALDI molecular imaging for clinical proteomic research: current state and fields of application

MALDI imaging mass spectrometry ('MALDI imaging') is an increasingly recognized technique for biomarker research. After years of method development in the scientific community, the technique is now increasingly applied in clinical research. In this article, we discuss the use of MALDI imaging in clinical proteomics and put it in context with classical proteomics techniques. We also highlight a number of upcoming challenges for personalized medicine, development of targeted therapies and diagnostic molecular pathology where MALDI imaging could help.     

纳米金在分子影像学中的应用进展

分子影像学的出现将传统的以解剖结构为成像基础的医学影像学带入到以图像阐释细胞／分子结构和功能以及病理改变的新时代。伴随着“后基因组”时代的到来以及“个体化医疗”的兴起,分子影像学对医学领域带来了里程碑式的革命并日益发挥重要作用。在分子影像领域,寻找最佳的分子影像探针／对比剂以及成像方法,以获取更多的细胞或者分子的功能及病理改变的信息日益成为热门的研究领域。纳米金籍其自身的优点在分子影像学的发展中展示出日益广阔的前景。本文就分子影像学的相关技术及纳米金在分子影像学中的应用进展作一简要综述。     

纳米金在分子影像学中的应用进展

分子影像学的出现将传统的以解剖结构为成像基础的医学影像学带入到以图像阐释细胞/分子结构和功能以及病理改变的新时代。伴随着"后基因组"时代的到来以及"个体化医疗"的兴起,分子影像学对医学领域带来了里程碑式的革命并日益发挥重要作用。在分子影像领域,寻找最佳的分子影像探针/对比剂以及成像方法,以获取更多的细胞或者分子的功能及病理改变的信息日益成为热门的研究领域。纳米金籍其自身的优点在分子影像学的发展中展示出日益广阔的前景。本文就分子影像学的相关技术及纳米金在分子影像学中的应用进展作一简要综述。     

Recent findings from the human proteome project: opening the mass spectrometry toolbox to advance cancer diagnosis,surveillance and treatment

The Human Proteome Project stands to eclipse the Human Genome Project in terms of scope, content and interpretation. Its outputs, in conjunction with recent developments across the proteomics community, provide new tools for cancer research with the potential of providing clinically relevant insights into the disease. These collectively may guide the development of future diagnosis, surveillance and treatment strategies. Having established a robust organizational framework within the international community, the Human Proteome Organization and the proteomics community at large have made significant advances in biomarker discovery, detection, molecular imaging and in exploring tumor heterogeneity. Here, the authors discuss some developments in cancer proteomics and how they can be implemented to reduce the global burden of the disease.     

Cell cycle: proteomics gives it a spin

The eukaryotic cell division cycle has been studied at the molecular level for over 30 years, most fruitfully in model organisms. In the past 5 years, developments in mass spectrometry-based proteomics have been applied to the study of protein interactions and post-translational modifications involving key cell cycle regulators such as cyclin-dependent kinases and the anaphase-promoting complex, as well as effectors such as centrosomes, the kinetochore and DNA replication forks. In addition, innovations in chemical biology, functional proteomics and bioinformatics have been employed to study the cell cycle at the proteome level. This review surveys the contributions of proteomics to cell cycle research. The near future should see the application of more quantitative proteomic approaches to probe the dynamic aspects of the molecular system that underlie the cell cycle in model organisms and in human cells.     

Cell cycle: proteomics gives it a spin

The eukaryotic cell division cycle has been studied at the molecular level for over 30 years, most fruitfully in model organisms. In the past 5 years, developments in mass spectrometry-based proteomics have been applied to the study of protein interactions and post-translational modifications involving key cell cycle regulators such as cyclin-dependent kinases and the anaphase-promoting complex, as well as effectors such as centrosomes, the kinetochore and DNA replication forks. In addition, innovations in chemical biology, functional proteomics and bioinformatics have been employed to study the cell cycle at the proteome level. This review surveys the contributions of proteomics to cell cycle research. The near future should see the application of more quantitative proteomic approaches to probe the dynamic aspects of the molecular system that underlie the cell cycle in model organisms and in human cells.     

Thyroid tumors: novel insights from proteomic studies

Several genomics-based techniques have been applied in the last decade to the molecular characterization of cancer, which has led to a variety of applications suitable for improved diagnosis, prognosis and prediction of outcome to treatment. Proteomics-based approaches have also been seen as crucial to the discovery of biomarkers for early diagnosis and prognosis of tumors, as well as for a better understanding of the molecular bases of cancer. Accordingly, proteomic techniques have been used extensively for a better molecular characterization of thyroid tumors. In this field, three main directions have been preceded: first, proteomic studies of model systems; second, proteomics of thyroid tumor specimens; and third, serum proteomics. In this review, we describe the most relevant results that have been obtained for tumors derived from thyroid follicular cells using various proteomic approaches.     

Aptamer-based molecular imaging

Molecular imaging has greatly advanced basic biology and translational medicine through visualization and quantification of single/multiple molecular events temporally and spatially in a cellular context and in living organisms. Aptamers, short single-stranded nucleic acids selected in vitro to bind a broad range of target molecules avidly and specifically, are ideal molecular recognition elements for probe development in molecular imaging. This review summarizes the current state of aptamer-based biosensor development (probe design and imaging modalities) and their application in imaging small molecules, nucleic acids and proteins mostly in a cellular context with some animal studies. The article is concluded with a brief discussion on the perspective of aptamer-based molecular imaging.     

Computational proteomics of biomolecular interactions in the sequence and structure space of the tyrosine kinome: deciphering the molecular basis of the kinase inhibitors selectivity

Understanding and predicting the molecular basis of protein kinases specificity against existing therapeutic agents remains highly challenging and deciphering this complexity presents an important problem in discovery and development of effective cancer drugs. We explore a recently introduced computational approach for in silico profiling of the tyrosine kinases binding specificity with a class of the pyrido-[2,3-d]pyrimidine kinase inhibitors. Computational proteomics analysis of the ligand-protein interactions using parallel simulated tempering with an ensemble of the tyrosine kinases crystal structures reveals an important molecular determinant of the kinase specificity. The pyrido-[2,3-d]pyrimidine inhibitors are capable of dynamically interacting with both active and inactive forms of the tyrosine kinases, accommodating structurally different kinase conformations with a similar binding affinity. Conformational tolerance of the protein tyrosine kinases binding with the pyrido[2,3-d]pyrimidine inhibitors provides the molecular basis for the broad spectrum of potent activities and agrees with the experimental inhibition profiles. The analysis of the pyrido[2,3-d]pyrimidine sensitivities against a number of clinically relevant ABL kinase mutants suggests an important role of conformational adaptability of multitargeted kinase inhibitors in developing drug resistance mechanisms. The presented computational approach may be useful in complementing proteomics technologies to characterize activity signatures of small molecules against a large number of potential kinase targets.     

Proteomics in Alzheimer's disease: insights into potential mechanisms of neurodegeneration

Proteomics involves the identification of unknown proteins following their separation, often using two-dimensional electrophoresis, digestion of particular proteins of interest by trypsin, determination of the molecular weight of the resulting peptides, and database searching to make the identification of the proteins. Application of proteomics to Alzheimer's disease (AD), the major dementing disorder of the elderly, has just begun. Differences in protein expression and post-translational modification (mostly oxidative modification) of proteins from AD brain and peripheral tissue, as well as in brain from rodent models of AD, have yielded insights into potential molecular mechanisms of neurodegeneration in this dementing disorder. This review surveys the proteomics studies relevant to AD, from which new understandings of the pathology, biochemistry, and physiology of AD are beginning to emerge.     

超声分子成像：机遇与挑战

随着分子生物学、蛋白组学、基因组学、计算机工程学等学科的不断进步,交叉融合,分子成像逐渐登上历史的舞台,成为研究热点。而超声分子成像随之迅猛发展,近年来超声微泡制备技术的成熟和超声造影检查技术的不断进步,超声造影不再局限获取组织的血流灌注信息,而是逐渐成为特异性的超声分子成像。目前使用超声对比剂研究分子成像和靶向治疗仍处于初级阶段。但是,各种分子成像技术的不断革新和发展,超声分子成像面临着重大的挑战,而在挑战背后同样面临着难逢的机遇。超声医学和分子生物学的迅猛发展,超声分子成像必将成为诊断和治疗疾病的新的手段和方法。超声造影剂仍有许多未能解决的问题,像如何延长微泡的半衰期、如何增强微泡的敏感性和特异性,如何增强目的基因的表达,如何处理组织损伤和高频超声之间的关系等问题,但是如果能解决这些问题,超声造影在现代医学的诊断和治疗中将起到重要的作用。现将超声分子成像综述如下。     

Tagging and detection strategies for activity-based proteomics

The field of activity-based proteomics is a relatively new discipline that makes use of small molecules, termed activity-based probes (ABPs), to tag and monitor distinct sets of proteins within a complex proteome. These activity-dependant labels facilitate analysis of systems-wide changes at the level of enzyme activity rather than simple protein abundance. While the use of small molecule inhibitors to label enzyme targets is not a new concept, the past ten years have seen a rapid expansion in the diversity of probe families that have been developed. In addition to increasing the number and types of enzymes that can be targeted by this method, there has also been an increase in the number of methods used to visualize probes once they are bound to target enzymes. In particular, the use of small organic fluorophores has created a wealth of applications for ABPs that range from biochemical profiling of diverse proteomes to direct imaging of active enzymes in live cells and even whole animals. In addition, the advent of new bioorthogonal coupling chemistries now enables a diverse array of tags to be added after targets are labeled with an ABP. This strategy has opened the door to new in vivo applications for activity-based proteomic methods.     

Tissue imaging using MALDI-MS: a new frontier of histopathology proteomics

Modern pathology is an amalgam of many disciplines, such as microbiology, biochemistry and immunology, which historically have been intermingled with the practice of clinical medicine. For centuries, the pre-eminent pathological tool, at least in the context of patients, was a post-mortem examination. With the advent of optical microscopes, morphology became a predominant means of developing tissue classification. A further paradigm shift occurred in the attempt to understand the nature and origin of disease; the recognition that, ultimately, it is the derangement in the structure and function of genes and proteins that causes human disease. More recent progress in pathology has led to the use of genomics and molecular technologies, including DNA sequencing, microarray analysis, PCR, in situ hybridization and proteomics. Today, the newest frontier appears to be histopathology proteomics, which adds the mass spectrometer to the arsenal of tools for the direct analysis of tissue biopsies and molecular diagnosis. Typically called MALDI imaging, this technique takes mass spectral snapshots of intact tissue slices, revealing how proteins and peptides are spatially distributed within a given sample. In this review, MALDI imaging technology is presented as well as applications of such technology in cancer or neurodegenerative diseases.     

Tissue imaging using MALDI-MS: a new frontier of histopathology proteomics

Modern pathology is an amalgam of many disciplines, such as microbiology, biochemistry and immunology, which historically have been intermingled with the practice of clinical medicine. For centuries, the pre-eminent pathological tool, at least in the context of patients, was a post-mortem examination. With the advent of optical microscopes, morphology became a predominant means of developing tissue classification. A further paradigm shift occurred in the attempt to understand the nature and origin of disease; the recognition that, ultimately, it is the derangement in the structure and function of genes and proteins that causes human disease. More recent progress in pathology has led to the use of genomics and molecular technologies, including DNA sequencing, microarray analysis, PCR, in situ hybridization and proteomics. Today, the newest frontier appears to be histopathology proteomics, which adds the mass spectrometer to the arsenal of tools for the direct analysis of tissue biopsies and molecular diagnosis. Typically called MALDI imaging, this technique takes mass spectral snapshots of intact tissue slices, revealing how proteins and peptides are spatially distributed within a given sample. In this review, MALDI imaging technology is presented as well as applications of such technology in cancer or neurodegenerative diseases.     

Matrix-assisted laser desorption/ionization imaging mass spectrometry: a promising technique for reproductive research

Matrix-assisted laser desorption/ionization (MALDI) tissue imaging mass spectrometry is particularly promising among the numerous applications of mass spectrometry. It is used for probing and analyzing the spatial arrangement of a wide range of molecules, including proteins, peptides, lipids, drugs, and metabolites, directly in thin slices of tissue. In the field of proteomics, the technology avoids tedious and time-consuming extraction and fractionation steps classically required for sample analysis. MALDI imaging mass spectrometry is increasingly recognized as a powerful method for clinical proteomics, particularly in cancer research. The technology has particular potential for the discovery of new tissue biomarker candidates, classification of tumors, early diagnosis or prognosis, elucidating pathogenesis pathways, and therapy monitoring. Over recent years, MALDI imaging mass spectrometry has been used for molecular profiling and imaging directly in male and female reproductive tissues. This review will consider some of the recent publications in the field, addressing a range of issues covering embryo development, gene expression product profiling during gametogenesis, and seeking and identifying biomarkers of reproductive cancers. The wealth of advances in mass spectrometry imaging will inevitably attract biologists and clinicians as the advantages and power of this technology become more widely known. This review will also discuss bottlenecks and the many technical issues that remain to be resolved before laboratories in the field can adopt the technology. We foresee that MALDI imaging mass spectrometry will have a major impact in reproductive research by opening new avenues to the understanding of various molecular mechanisms and the diagnosis of reproductive pathologies.     

High-resolution imaging using a novel atomic force microscope and confocal laser scanning microscope hybrid instrument: essential sample preparation aspects

The recent data explosion in global gene expression profiling and proteomics has resulted in a need to determine the mechanistic role of biomarker signatures in pathogenicity. Consequently, elaborate technologies are required to assess increasingly smaller sub-cellular compartments and constituents. We describe the development, evaluation and application of an efficient sample preparation methodology to facilitate coupled atomic force microscopy and confocal laser scanning microscopy (AFM–CLSM), providing a novel means of concurrent high-resolution structural and fluorescence imaging. Due to their fragile nature and nanoscale dimensions, filopodia were selected as a model to develop the procedure that maximised fluorescence response, while maintaining epithelial cell ultra-structure. Fixation with ultra-pure methanol-free formaldehyde coupled to quantum dot nanocrystal labelling proved to be vital in achieving high quality AFM–CLSM images. We demonstrated for the first time that filopodia have a “quilted” surface structure. Additionally, high ultra-structural ridges on the apical cell surface resolved by AFM corresponded to punctate moesin clusters, representing direct visualisation of moesin linkages between transmembrane proteins and the cytoskeleton. The capacity of this novel multi-modal imaging technique to probe topography, molecular composition and biophysical properties of ultra-structural features therefore provides unique information that will significantly contribute to our understanding of cellular structure–function relationships.     

Current strategies and findings in clinically relevant post-translational modification-specific proteomics

Mass spectrometry-based proteomics has considerably extended our knowledge about the occurrence and dynamics of protein post-translational modifications (PTMs). So far, quantitative proteomics has been mainly used to study PTM regulation in cell culture models, providing new insights into the role of aberrant PTM patterns in human disease. However, continuous technological and methodical developments have paved the way for an increasing number of PTM-specific proteomic studies using clinical samples, often limited in sample amount. Thus, quantitative proteomics holds a great potential to discover, validate and accurately quantify biomarkers in body fluids and primary tissues. A major effort will be to improve the complete integration of robust but sensitive proteomics technology to clinical environments. Here, we discuss PTMs that are relevant for clinical research, with a focus on phosphorylation, glycosylation and proteolytic cleavage; furthermore, we give an overview on the current developments and novel findings in mass spectrometry-based PTM research.     

Proteomics in the study of hippocampal plasticity

Synaptic plasticity is the dynamic regulation of the strength of synaptic communication between nerve cells. It is central to neuronal development as well as experience-dependent remodeling of the adult nervous system as occurs during memory formation. Aberrant forms of synaptic plasticity also accompany a variety of neurological and psychiatric diseases, and unraveling the biological basis of synaptic plasticity has been a major goal in neurobiology research. The biochemical and structural mechanisms underlying different forms of synaptic plasticity are complex, involving multiple signaling cascades, reconfigurations of structural proteins and the trafficking of synaptic proteins. As such, proteomics should be a valuable tool in dissecting the molecular events underlying normal and disease-related forms of plasticity. In fact, progress in this area has been disappointingly slow. We discuss the particular challenges associated with proteomic interrogation of synaptic plasticity processes and outline ways in which we believe proteomics may advance the field over the next few years. We pay particular attention to technical advances being made in small sample proteomics and the advent of proteomic imaging in studying brain plasticity.