Novel proteomic approaches for tissue analysis

Proteomics, the global study of protein expression and characteristics, has recently emerged as a key component in the field of molecular analysis. The dynamic nature of proteins, from ion channels to chaperones, presents a challenge, yet the understanding of these molecules provides a rich source of information. When applying proteomic analysis directly to human tissue samples, additional difficulties arise. The following article presents an overview of the current proteomic tools used in the analysis of tissues, beginning with conventional methods such as western blot analysis and 2D polyacrylamide gel electrophoresis. The most current high-throughput techniques being used today are also reviewed. These include protein arrays, reverse-phase protein lysate arrays, matrix-assisted laser desorption/ionization, surface-enhanced laser desorption/ionization and layered expression scanning. In addition, bioinformatics as well as issues regarding tissue preservation and microdissection to obtain pure cell populations are included. Finally, future directions of the tissue proteomics field are discussed.     

Proteomic analysis of tissue samples in translational breast cancer research

In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis, and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies – based on the analysis of clinically relevant samples – that promise to accelerate the translation of basic discoveries into the daily breast cancer clinical practice. In particular, we address major issues in experimental design by reviewing the strengths and weaknesses of current proteomic strategies in the context of the analysis of human breast tissue specimens.     

Optimal molecular profiling of tissue and tissue components

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.     

Analysis conditions for proteomic profiling of mammalian tissue and cell extracts with antibody microarrays

Antibody microarrays are a developing tool for global proteomic profiling. A protocol was established that permits robust analyses of protein extracts from mammalian tissues and cells rather than body fluids. The factors optimized were buffer composition for surface blocking, blocking duration, protein handling and processing, labeling parameters like type of dye, molar ratio of label versus protein, and dye removal, as well as incubation parameters such as duration, temperature, buffer, and sample agitation.     

A prostate cancer tissue specific spectral library for targeted proteomic analysis

Prostate cancer is the most common cancer in males worldwide. Mass spectrometry-based targeted proteomics has demonstrated great potential in quantifying proteins from formalin-fixed paraffin-embedded (FFPE) and (fresh) frozen biopsy tissues. Here we provide a comprehensive tissue-specific spectral library for targeted proteomic analysis of prostate tissue samples. Benign and malignant FFPE prostate tissue samples were processed into peptide samples by pressure cycling technology (PCT)-assisted sample preparation, and fractionated with high-pH reversed phase liquid chromatography (RPLC). Based on data-dependent acquisition (DDA) MS analysis using a TripleTOF 6600, we built a library containing 108,533 precursors, 84,198 peptides and 9384 unique proteins (1% FDR). The applicability of the library was demonstrated in prostate specimens.     

Targeted protein-omic methods are bridging the gap between proteomic and hypothesis-driven protein analysis approaches

While proteomic methods have illuminated many areas of biological protein space, many fundamental questions remain with regard to systems-level relationships between mRNAs, proteins and cell behaviors. While mass spectrometric methods offer a panoramic picture of the relative expression and modification of large numbers of proteins, they are neither optimal for the analysis of predefined targets across large numbers of samples nor for assessing differences in proteins between individual cells or cell compartments. Conversely, traditional antibody-based methods are effective at sensitively analyzing small numbers of proteins across small numbers of conditions, and can be used to analyze relative differences in protein abundance and modification between cells and cell compartments. However, traditional antibody-based approaches are not optimal for analyzing large numbers of protein abundances and modifications across many samples. In this article, we will review recent advances in methodologies and philosophies behind several microarray-based, intermediate-level, ‘protein-omic’ methods, including a focus on reverse-phase lysate arrays and micro-western arrays, which have been helpful for bridging gaps between large- and small-scale protein analysis approaches and have provided insight into the roles that protein systems play in several biological processes.     

Breast tumor metastasis: analysis via proteomic profiling

The ability to predict the metastatic behavior of a patient’s cancer, as well as to detect and eradicate such recurrences, remain major clinical challenges in oncology. While many potential molecular biomarkers have been identified and tested previously, none have greatly improved the accuracy of specimen evaluation over routine histopathological criteria and, to date, they predict individual outcomes poorly. The ongoing development of high-throughput proteomic profiling technologies is opening new avenues for the investigation of cancer and, through application in tissue-based studies and animal models, will facilitate the identification of molecular signatures that are associated with breast tumor cell phenotype. The appropriate use of these approaches has the potential to provide efficient biomarkers, and to improve our knowledge of tumor biology. This, in turn, will enable the development of targeted therapeutics aimed at ameliorating the lethal dissemination of breast cancer. In this review, we focus on the accumulating proteomic signatures of breast tumor progression, particularly those that correlate with the occurrence of distant metastases, and discuss some of the expected future developments in the field.     

New approaches towards integrated proteomic databases and depositories

Since the publication of the human genome, two key points have emerged. First, it is still not certain which regions of the genome code for proteins. Second, the number of discrete protein-coding genes is far fewer than the number of different proteins. Proteomics has the potential to address some of these postgenomic issues if the obstacles that we face can be overcome in our efforts to combine proteomic and genomic data. There are many challenges associated with high-throughput and high-output proteomic technologies. Consequently, for proteomics to continue at its current growth rate, new approaches must be developed to ease data management and data mining. Initiatives have been launched to develop standard data formats for exchanging mass spectrometry proteomic data, including the Proteomics Standards Initiative formed by the Human Proteome Organization. Databases such as SwissProt and Uniprot are publicly available repositories for protein sequences annotated for function, subcellular location and known potential post-translational modifications. The availability of bioinformatics solutions is crucial for proteomics technologies to fulfil their promise of adding further definition to the functional output of the human genome. The aim of the Oxford Genome Anatomy Project is to provide a framework for integrating molecular, cellular, phenotypic and clinical information with experimental genetic and proteomics data. This perspective also discusses models to make the Oxford Genome Anatomy Project accessible and beneficial for academic and commercial research and development.     

Comprehensive proteomic analysis of Penicillium verrucosum

Mass spectrometric identification of proteins in species lacking validated sequence information is a major problem in veterinary science. In the present study, we used ochratoxin A producing Penicillium verrucosum to identify and quantitatively analyze proteins of an organism with yet no protein information available. The work presented here aimed to provide a comprehensive protein identification of P. verrucosum using shotgun proteomics. We were able to identify 3631 proteins in an “ab initio” translated database from DNA sequences of P. verrucosum. Additionally, a sequential window acquisition of all theoretical fragment‐ion spectra analysis was done to find differentially regulated proteins at two different time points of the growth curve. We compared the proteins at the beginning (day 3) and at the end of the log phase (day 12).     

Proteomic approaches for studying human parenchymal lung diseases

Parenchymal lung diseases comprise a wide variety of diseases, with different etiologies, pathogeneses and prognoses. This perspective provides an overview of two different disease types: chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Chronic obstructive pulmonary disease, which is related to smoking, is one of the leading causes of chronic morbidity and mortality around the world, being characterized by airway obstruction and parenchymal lung damage (emphysema). Idiopathic pulmonary fibrosis of unknown etiology is classified as one of the most important idiopathic interstitial pneumonias and is connected to patchy but progressive lung fibrosis. Both diseases are generally diagnosed late and respond poorly to medical therapies. Although numerous biomarkers have been proposed for these diseases, they have not been validated or implemented into clinical practice. This perspective emphasizes some typical features of these diseases with different types of lung damage, how they are reflected in different samples, as well as potential advances and problems of current and future nonbiased proteomic approaches.     

A simple,chisel-assisted mechanical microdissection system for harvesting homogenous plant tissue suitable for the analysis of nucleic acids and proteins

Many physiological processes are limited to specific tissues or even specific cell types. Analysing entire plants or organs results in averaged data of all cell types contained in the sample; thus, specific metabolic functions cannot be assigned to individual cell types. A higher spatial resolution is required. By microdissecting plant organs, homogeneous material can be obtained. If a suitable amount of material is collected, standard analytical methods can be applied to elucidate cell type-specific processes. The collection of sufficient quantities of homogeneous material can be done by means of mechanical microdissection. This technique is a low-cost alternative to laser-coupled microdissection techniques. Here we describe a protocol for chisel-assisted mechanical microdissection of embedded plant material and demonstrate that the collected material is suitable to obtain nucleic acids and proteins.     

蛋白质N末端组学研究进展

蛋白质的N末端作为合成的起始,其氨基酸序列组成及翻译后修饰直接影响着蛋白质的活性、稳定性和细胞内定位,调控着细胞内的信号转导,甚至决定了这些蛋白质的命运。对蛋白质N末端组学的系统研究不仅可以揭示N末端区域对整个蛋白质的重要作用,有助于我们深入地了解蛋白质在各种生命活动中所扮演的角色,同时在实现蛋白质组高覆盖、基因组重注释等方面也有着重要的价值。本文结合我们的现有工作,综述了近年来蛋白质N末端组学的研究进展,尤其是一些重要的基于质谱的N末端富集技术和方法。     

Unravelling the biology of chromatin in health and cancer using proteomic approaches

Introduction: Chromatin remodeling complexes play important roles in the control of genome regulation in both normal and diseased states, and are therefore critical components for the regulation of epigenetic states in cells. Given the role epigenetics plays in cancer, for example, chromatin remodeling complexes are routinely targeted for therapeutic intervention.

Areas covered: Protein mass spectrometry and proteomics are powerful technologies used to study and understand chromatin remodeling. While impressive progress has been made in this area, there remain significant challenges in the application of proteomic technologies to the study of chromatin remodeling. As parts of large multi-subunit complexes that can be heavily modified with dynamic post-translational modifications, challenges in the study of chromatin remodeling complexes include defining the content, determining the regulation, and studying the dynamics of the complexes under different cellular states.

Expert commentary: Impwortant considerations in the study of chromatin remodeling complexes include the complexity of sample preparation, the choice of proteomic methods for the analysis of samples, and data analysis challenges. Continued research in these three areas promise to yield even greater insights into the biology of chromatin remodeling and epigenetics and the dynamics of these systems in human health and cancer.     

Effect of Immunohistochemistry on Molecular Analysis of Tissue Samples: Implications for Microdissection Technologies

Laser-based tissue microdissection is an important tool for the molecular evaluation of histological sections. The technology has continued to advance since its initial commercialization in the 1990s, with improvements in many aspects of the process. More recent developments are tailored toward an automated, operator-independent mode that relies on antibodies as targeting probes, such as immuno–laser capture microdissection or expression microdissection (xMD). Central to the utility of expression-based dissection techniques is the effect of the staining process on the biomolecules in histological sections. To investigate this issue, the authors analyzed DNA, RNA, and protein in immunostained, microdissected samples. DNA was the most robust molecule, exhibiting no significant change in quality after immunostaining but a variable 50% to 75% decrease in the total yield. In contrast, RNA in frozen and ethanol-fixed, paraffin-embedded samples was susceptible to hydrolysis and digestion by endogenous RNases during the initial steps of staining. Proteins from immunostained tissues were successfully analyzed by one-dimensional electrophoresis and mass spectrometry but were less amenable to solution phase assays. Overall, the results suggest investigators can use immunoguided microdissection methods for important analytic techniques; however, continued improvements in staining protocols and molecular extraction methods are key to further advancing the capability of these methods.     

Data merging for integrated microarray and proteomic analysis.

The functioning of even a simple biological system is much more complicated than the sum of its genes, proteins and metabolites. A premise of systems biology is that molecular profiling will facilitate the discovery and characterization of important disease pathways. However, as multiple levels of effector pathway regulation appear to be the norm rather than the exception, a significant challenge presented by high-throughput genomics and proteomics technologies is the extraction of the biological implications of complex data. Thus, integration of heterogeneous types of data generated from diverse global technology platforms represents the first challenge in developing the necessary foundational databases needed for predictive modelling of cell and tissue responses. Given the apparent difficulty in defining the correspondence between gene expression and protein abundance measured in several systems to date, how do we make sense of these data and design the next experiment? In this review, we highlight current approaches and challenges associated with integration and analysis of heterogeneous data sets, focusing on global analysis obtained from high-throughput technologies.     

Utility of reverse phase protein arrays: applications to signalling pathways and human body arrays.

Protein microarrays offer a new means by which to conduct quantitative profiling of disease-associated proteins. The knowledge gained may provide novel strategies for early detection, diagnosis and therapeutic intervention. A variety of sophisticated approaches, including gene arrays, sequencing consortiums and large-scale two-dimensional gel electrophoresis, continue to generate lists of proteins potentially linked to disease aetiology and progression. The challenge is to evaluate quantitatively promising lead protein candidates using matched normal and diseased cell populations. In contrast to the antibody array, the reverse phase protein microarrays (RPPA) do not require labelling of cellular protein lysates, and constitute a sensitive high throughput platform for marker screening, pathophysiology investigation and therapeutic monitoring. In this paper, examples will be provided using RPPAs in the study of the apoptotic signalling cascade and in the evaluation of the expression of organ-specific protein makers using microdissected human organ cell lysates configured as 'human body arrays'.     

An optimized procedure for the capture,fractionation and proteomic analysis of proteins using hydrogel nanoparticles

We have developed an optimized procedure using dual size exclusion/affinity hydrogel nanoparticles to capture and comparatively analyze low molecular mass proteins directly from biological samples. The method described facilitates charge‐ and size‐dependent protein binding, direct analysis by MS or other means and is highly reproducible. A comparative analysis of the low molecular mass proteome of plasma following freeze–thaw immediately after venipuncture is used to illustrate proof‐of‐concept. The technique described is rapid and may be easily reproduced in any laboratory.     

Mass spectrometry-based proteomic approaches to study pathogenic bacteria-host interactions

Elucidation of molecular mechanisms underlying hostpathogen interactions is important for control and treatment of infectious diseases worldwide. Within the last decade, mass spectrometry (MS)-based proteomics has become a powerful and effective approach to better understand complex and dynamic host-pathogen interactions at the protein level. Herein we will review the recent progress in proteomic analyses towards bacterial infection of their mammalian host with a particular focus on enteric pathogens. Large-scale studies of dynamic proteomic alterations during infection will be discussed from the perspective of both pathogenic bacteria and host cells.