Postgenomics of Neisseria meningitidis for vaccines development

Meningococcal disease is a global problem. Multivalent (A, C, Y, W135) conjugate vaccines have been developed and licensed; however, an effective vaccine against serogroup B has not yet become available. Outer membrane vesicle (OMV) vaccines have been used to disrupt serogroup B epidemics and outbreaks. Postgenomic technologies have been useful in aiding the discovery of new protein vaccine candidates. Moreover, proteomic technologies enable large-scale identification of membrane and surface-associated proteins, and provide suitable methods to characterize and standardize the antigen composition of OMV-based vaccines.     

Analysis of organelles within the nervous system: impact on brain and organelle functions

Currently, neuroproteomic approaches aimed at the profiling of total brain areas generally mirror the expression of the most abundant proteins, but fail to uncover less abundant proteins. By contrast, the focus on typical brain subproteomes, (e.g., synaptic vesicles, synaptic terminal membranes or the postsynaptic density), may give a more specific insight into brain function. Subproteomes are accessible via several strategies, including subcellular fractionation or affinity-based pull-down approaches. Combined with mass spectrometric quantification approaches, subcellular proteomics is expected to reveal differences in the protein constitution of related cellular organelles. Focusing on novel functions and mechanistic models, we review recent data on the analysis of brain-derived organelles and subproteomes, including presynaptic termini, synaptic vesicles, neuronal plasma membranes, postsynaptic density and neuromelanin granules, which were identified as novel lysosome-related organelles within the human brain.     

Oncoproteomics: current trends and future perspectives

Oncoproteomics is the application of proteomics technologies in oncology. Functional proteomics is a promising technique for the rational identification of biomarkers and novel therapeutic targets for cancers. Recent progress in proteomics has opened new avenues for tumor-associated biomarker discovery. With the advent of new and improved proteomics technologies, such as the development of quantitative proteomic methods, high-resolution, -speed and -sensitivity mass spectrometry and protein arrays, as well as advanced bioinformatics for data handling and interpretation, it is now possible to discover biomarkers that can reliably and accurately predict outcomes during cancer management and treatment. However, there are several difficulties in the study of proteins/peptides that are not inherent in the study of nucleic acids. New challenges arise in large-scale proteomic profiling when dealing with complex biological mixtures. Nevertheless, oncoproteomics offers great promise for unveiling the complex molecular events of tumorigenesis, as well as those that control clinically important tumor behaviors, such as metastasis, invasion and resistance to therapy. In this review, the development and advancement of oncoproteomics technologies for cancer research in recent years are expounded.     

Chemical proteomics from a nuclear magnetic resonance spectroscopy perspective

Proteomics is the study of the protein complement of a genome and employs a number of newly emerging tools. One such tool is chemical proteomics, which is a branch of proteomics devoted to the exploration of protein function using both in vitro and in vivo chemical probes. Chemical proteomics aims to define protein function and mechanism at the level of directly observed protein–ligand interactions, whereas chemical genomics aims to define the biological role of a protein using chemical knockouts and observing phenotypic changes. Chemical proteomics is therefore traditional mechanistic biochemistry performed in a systems-based manner, using either activity- or affinity-based probes that target proteins related by chemical reactivities or by binding site shape/properties, respectively. Systems are groups of proteins related by metabolic pathway, regulatory pathway or binding to the same ligand. Studies can be based on two main types of proteome samples: pooled proteins (1 mixture of N proteins) or isolated proteins in a given system and studied in parallel (N single protein samples). Although the field of chemical proteomics originated with the use of covalent labeling strategies such as isotope-coded affinity tagging, it is expanding to include chemical probes that bind proteins noncovalently, and to include more methods for observing protein–ligand interactions. This review presents an emerging role for nuclear magnetic resonance spectroscopy in chemical proteomics, both in vitro and in vivo. Applications include: functional proteomics using cofactor fingerprinting to assign proteins to gene families; gene family-based structural characterizations of protein–ligand complexes; gene family-focused design of drug leads; and chemical proteomic probes using nuclear magnetic resonance SOLVE and studies of protein–ligand interactions in vivo.     

Tsetse flies,trypanosomes, humans and animals: what is proteomics revealing about their crosstalks?

Human and animal African trypanosomoses, or sleeping sickness and Nagana, are neglected vector-borne parasitic diseases caused by protozoa belonging to the Trypanosoma genus. Advances in proteomics offer new tools to better understand host–vector–parasite crosstalks occurring during the complex parasitic developmental cycle, and to determine the outcome of both transmission and infection. In this review, we summarize proteomics studies performed on African trypanosomes and on the interactions with their vector and mammalian hosts. We discuss the contributions and pitfalls of using diverse proteomics tools, and argue about the interest of pathogenoproteomics, both to generate advances in basic research on the best knowledge and understanding of host–vector–pathogen interactions, and to lead to the concrete development of new tools to improve diagnosis and treatment management of trypanosomoses in the near future.     

2D-LC/MS techniques for the identification of proteins in highly complex mixtures

Today, 2D online or offline liquid chromatography/mass spectrometry is state of the art for the identification of proteins from complex proteome samples in many laboratories. Both 2D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method. The high-performance liquid chromatography techniques complement the 2D-gel techniques supporting their weaknesses. This is especially true for the gel separation of hydrophobic membrane proteins, which play an important role in living cells as well as being important targets for future pharmaceutical drugs.     

High-throughput proteomics using Fourier transform ion cyclotron resonance mass spectrometry

The advent of high-throughput proteomic technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of the cellular machinery. Here, recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry are reviewed along with its potential application to high-throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system level.     

Current progress in protein profiling of complex samples

Accurate cancer biomarkers are needed for early detection, disease classification, prediction of therapeutic response and monitoring treatment. While there appears to be no shortage of candidate biomarker proteins, a major bottleneck in the biomarker pipeline continues to be their verification by enzyme linked immunosorbent assays. Multiple reaction monitoring (MRM), also known as selected reaction monitoring, is a targeted mass spectrometry approach to protein quantitation and is emerging to bridge the gap between biomarker discovery and clinical validation. Highly multiplexed MRM assays are readily configured and enable simultaneous verification of large numbers of candidates facilitating the development of biomarker panels which can increase specificity. This review focuses on recent applications of MRM to the analysis of plasma and serum from cancer patients for biomarker verification. The current status of this approach is discussed along with future directions for targeted mass spectrometry in clinical biomarker validation.     

Role of oncoproteomics in the personalized management of cancer

Oncoproteomics is the term used to describe the application of proteomic technologies in oncology and parallels the related field of oncogenomics. It is now contributing to the development of personalized management of cancer. Proteomic technologies are used for the identification of biomarkers in cancer, which will facilitate the integration of diagnosis and therapy of cancer. Molecular diagnostics, laser capture microdissection and protein biochips are among the technologies that are having an important impact on oncoproteomics. The discovery of protein patterns developed by the US Food and Drug Administration/National Cancer Institute Clinical Proteomics Program is capable of distinguishing cancer and disease-free states with high sensitivity and specificity and will also facilitate the development of personalized therapy of cancer. Examples of application are given for breast and prostate cancer and a selection of companies and their collaborations that are developing application of proteomics to personalized treatment of cancer are discussed. Continued refinement of techniques and methods to determine the abundance and status of proteins in vivo holds great promise for the future study of normal cells and the pathology of associated neoplasms. Personalized cancer therapy is expected to be in the clinic by the end of the first decade of the 21st century.     

News in Brief

Protein microarrays are versatile tools for parallel, miniaturized screening of binding events involving large numbers of immobilized proteins in a time- and cost-effective manner. They are increasingly applied for high-throughput protein analyses in many research areas, such as protein interactions, expression profiling and target discovery. While conventionally made by the spotting of purified proteins, recent advances in technology have made it possible to produce protein microarrays through in situ cell-free synthesis directly from corresponding DNA arrays. This article reviews recent developments in the generation of protein microarrays and their applications in proteomics and diagnostics.