Proteomics studies of post-translational modifications in plants

Post-translational modifications of proteins greatly increase protein complexity and dynamics, co-ordinating the intricate regulation of biological events. The global identification of post-translational modifications is a difficult task that is currently accelerated by advances in proteomics techniques. There has been significant development in sample preparation methods and mass spectrometry instrumentation. To reduce the complexity and to increase the amount of modified proteins available for analysis, proteins are usually subjected to prefractionation such as chromatographic purification and affinity enrichment. In this review, the post-translational modification studies in plants are summarized. The sample preparation strategies applied to each study are also described. These include affinity-based enrichment methods, immobilized metal affinity chromatography and immunoprecipitation used for phosphorylation and ubiquitination studies, respectively, and the phase partitioning approach for glycosylphosphatidylinositol modification studies.     

Mass spectrometry-based plasma proteomics: state of the art and future outlook

Mass spectrometry-based plasma proteomics is a field where intense research has been performed during the last decade. Being closely linked to biomarker discovery, the field has received a fair amount of criticism, mostly due to the low number of novel biomarkers reaching the clinic. However, plasma proteomics is under gradual development with improvements on fractionation methods, mass spectrometry instrumentation and analytical approaches. These recent developments have contributed to the revival of plasma proteomics. The goal of this review is to summarize these advances, focusing in particular on fractionation methods, both for targeted and global mass spectrometry-based plasma analysis.     

New developments in profiling and imaging of proteins from tissue sections by MALDI mass spectrometry

Molecular imaging of tissue by MALDI mass spectrometry is a powerful tool for visualizing the spatial distribution of constituent analytes with high molecular specificity. Although the technique is relatively young, it has already contributed to the understanding of many diverse areas of human health. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. The purpose of this review is to highlight some of the more recent technological advances that have improved the efficiency of imaging mass spectrometry for clinical applications. Advances in the way MALDI mass spectrometry is integrated with histology, improved methods for automation, and better tools for data analysis are outlined in this review. Refined top-down strategies for the identification and validation of candidate biomarkers found in tissue sections are discussed. A clinical example highlighting the application of these methods to a cohort of clinical samples is described.     

Proteomic analysis of cellular signaling

Protein phosphorylation events are key regulators of cellular signaling processes. In the era of functional genomics, rational drug design programs demand large-scale high-throughput analysis of signal transduction cascades. Significant improvements in the area of mass spectrometry-based proteomics have provided exciting opportunities for rapid progress toward global protein phosphorylation analysis. This review summarizes several recent advances made in the field of phosphoproteomics with an emphasis placed on mass spectrometry instrumentation, enrichment methods and quantification strategies. In the near future, these technologies will provide a tool that can be used for quantitative investigation of signal transduction pathways to generate new insights into biologic systems.     

Proteomic analysis of cellular signaling

Protein phosphorylation events are key regulators of cellular signaling processes. In the era of functional genomics, rational drug design programs demand large-scale high-throughput analysis of signal transduction cascades. Significant improvements in the area of mass spectrometry-based proteomics have provided exciting opportunities for rapid progress toward global protein phosphorylation analysis. This review summarizes several recent advances made in the field of phosphoproteomics with an emphasis placed on mass spectrometry instrumentation, enrichment methods and quantification strategies. In the near future, these technologies will provide a tool that can be used for quantitative investigation of signal transduction pathways to generate new insights into biologic systems.     

Metabolomic strategies for the identification of new enzyme functions and metabolic pathways

Recent technological advances in accurate mass spectrometry and data analysis have revolutionized metabolomics experimentation. Activity-based and global metabolomic profiling methods allow simultaneous and rapid screening of hundreds of metabolites from a variety of chemical classes, making them useful tools for the discovery of novel enzymatic activities and metabolic pathways. By using the metabolome of the relevant organism or close species, these methods capitalize on biological relevance, avoiding the assignment of artificial and non-physiological functions. This review discusses state-of-the-art metabolomic approaches and highlights recent examples of their use for enzyme annotation, discovery of new metabolic pathways, and gene assignment of orphan metabolic activities across diverse biological sources.     

Alternative polyadenylation: New insights from global analyses

Recent studies have revealed widespread mRNA alternative polyadenylation (APA) in eukaryotes and its dynamic spatial and temporal regulation. APA not only generates proteomic and functional diversity, but also plays important roles in regulating gene expression. Global deregulation of APA has been demonstrated in a variety of human diseases. Recent exciting advances in the field have been made possible in a large part by high throughput analyses using newly developed experimental tools. Here I review the recent progress in global studies of APA and the insights that have emerged from these and other studies that use more conventional methods.     

Studies using specific biomarkers for human exposure assessment to exogenous and endogenous chemical agents.

The extent of formation of carcinogen adducts with DNA and protein may be used to assess the biologically effective dose of these carcinogens in the tissue under study. In normal human tissues, such carcinogen adducts arise in part from exposures to exogenous genotoxic compounds, although it has been shown that endogenously formed carcinogens also make a significant contribution to the observed DNA and protein damage. Although, highly sensitive analytical methods, such as immunoassay, 32P-postlabelling and mass spectrometry have been developed and successfully applied to measure carcinogen adducts, further methodological advances are making these methods more amenable to molecular epidemiological studies. Thus, the use of immunoslot blot assays allows a higher sample throughput for adduct quantification. Liquid chromatographic separations of adducts, either for their radiochemical detection following 32P-postlabelling or for their determination by mass spectrometry, improves the specificity and applicability of these techniques. In this review, the sensitivities and specificities of the analytical methods used for adduct detection are compared and the limitations of these methods described.     

Promise and progress in environmental genomics: a status report on the applications of gene expression-based microarray studies in ecologically relevant fish species

The advent of any new technology is typically met with great excitement. So it was a few years ago, when the combination of advances in sequencing technology and the development of microarray technology made measurements of global gene expression in ecologically relevant species possible. Many of the review papers published around that time promised that these new technologies would revolutionize environmental biology as they had revolutionized medicine and related fields. A few years have passed since these technological advancements have been made, and the use of microarray studies in non‐model fish species has been adopted in many laboratories internationally. Has the relatively widespread adoption of this technology really revolutionized the fields of environmental biology, including ecotoxicology, aquaculture and ecology, as promised? Or have these studies merely become a novelty and a potential distraction for scientists addressing environmentally relevant questions? In this review, the promises made in early review papers, in particular about the advances that the use of microarrays would enable, are summarized; these claims are compared to the results of recent studies to determine whether the forecasted changes have materialized. Some applications, as discussed in the paper, have been realized and have led to advances in their field, others are still under development.     

News in Brief

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.     

Mass spectrometry approaches to monitor protein-drug interactions

Recent advances in mass spectrometry-based approaches have enabled the investigation of drug-protein interactions in various ways including the direct detection of drug-target complexes, the examination of drug-induced changes in the target protein structure, and the monitoring of enzymatic target activity. Mass spectrometry-based proteomics methods also permit the unbiased analysis of changes in protein abundance and post-translational modifications induced by drug action. Finally, chemoproteomic affinity enrichment studies enable the deconvolution of drug targets under close to physiological conditions. This review provides an overview of current methods for the characterization of drug-target interactions by mass spectrometry and describes a protocol for chemoproteomic target binding studies using immobilized bioactive molecules.     

Chromatographic and related techniques for the determination of aromatic heterocyclic amines in foods

Some 20 years ago, Japanese scientists discovered a new group of highly toxic compounds, classified as heterocyclic aromatic amines, from broiled and grilled meat and fish products. Numerous studies have shown that most HAs are mutagenic and carcinogenic, and the safety of HA-containing foods has become a concern for the public. To date, more than 20 different mutagenic and/or carcinogenic heterocyclic amines have been identified in foods. This paper reviews the analysis of foods for HAs with 145 references. We survey some of the numerous methods available for the chromatographic analysis of heterocyclic amines and highlight the recent advances. We discuss chromatographic and related techniques, including capillary electrophoresis, and their coupling to mass spectrometry for the determination of these contaminants in foods. In addition, the review summarises data on the content of HAs in various cooked foods.     

Three-dimensional models of HDL apoA-I: implications for its assembly and function

The purpose of this review is to highlight recent advances toward the refinement of a three-dimensional structure for lipid-bound apolipoprotein A-I (apoA-I) on recombinant HDL. Recently, X-ray crystallography has yielded a new structure for full-length, lipid-free apoA-I. Although this approach has not yet been successful in solving the three-dimensional structure of lipid-bound apoA-I, analysis of the X-ray structures has been of immense help in the interpretation of structural data obtained from other methods that yield structural information. Recent studies emphasize the use of mass spectrometry to unambiguously identify cross-linked peptides or to quantify solvent accessibility using hydrogen-deuterium exchange. The combination of mass spectrometry, molecular modeling, molecular dynamic analysis, and small-angle X-ray diffraction has provided additional structural information on apoA-I folding that complements previous approaches.     

Investigation of post-translational modifications in type 2 diabetes

The investigation of post-translational modifications (PTMs) plays an important role for the study of type 2 diabetes. The importance of PTMs has been realized with the advancement of analytical techniques. The challenging detection and analysis of post-translational modifications is eased by different enrichment methods and by high throughput mass spectrometry based proteomics studies. This technology along with different quantitation methods provide accurate knowledge about the changes happening in disease conditions as well as in normal conditions. In this review, we have discussed PTMs such as phosphorylation, N-glycosylation, O-GlcNAcylation, acetylation and advanced glycation end products in type 2 diabetes which have been characterized by high throughput mass spectrometry based proteomics analysis.     

Quantitative phosphoproteomic analysis of signaling network dynamics

Protein phosphorylation mediated cellular signaling is a highly regulated, dynamic process that controls many aspects of cellular biology. Over the past few years many methods have been developed to quantify temporal dynamics of protein phosphorylation, including mass spectrometry, which can be applied in both an unbiased, discovery mode and in a targeted mode to monitor specific phosphorylation sites. Other methods, such as kinase activity assays and antibody microarrays, have been applied to quantify central nodes in the signaling network, yielding intriguing biological insights. This review provides a concise overview of the latest advances in the quantitative analysis of signaling dynamics including a brief commentary on the future of the field.     

Recent advances in sialic acid-focused glycomics

Recent emergences of glycobiology, glycotechnology and glycomics have been clarifying enormous roles of carbohydrates in biological recognition systems. For example, cell surface carbohydrates existing as glycoconjugates (glycolipids, glycoproteins and proteoglycans) play crucial roles in cell-cell communication, cell proliferation and differentiation, tumor metastasis, inflammatory response or viral infection. In particular, sialic acids (SAs) existing as terminal residues in carbohydrate chains on cell surface are involved in signal recognition and adhesion to ligands, antibodies, enzymes and microbes. In addition, plasma free SAs and sialoglycans have shown great potential for disease biomarker discovery. Therefore, the development of efficient analytical methods for structural and functional studies of SAs and sialylglycans are very important and highly demanded. The problems of SAs and sialylglycans analysis are vanishingly small sample amount, complicated and unstable structures, and complex mixtures. Nevertheless, in the past decade, mass spectrometry in combination with chemical derivatization and modern separation methodologies has become a powerful and versatile technique for structural analysis of SAs and sialylglycans. This review summarizes these recent advances in glycomic studies on SAs and sialylglycans. Specially, derivatization and capturing of SAs and sialylglycans combined with mass spectrometry analysis are highlighted.     

Dissecting the ubiquitin pathway by mass spectrometry

Protein modification by ubiquitin is a central regulatory mechanism in eukaryotic cells. Recent proteomics developments in mass spectrometry enable systematic analysis of cellular components in the ubiquitin pathway. Here, we review the advances in analyzing ubiquitinated substrates, determining modified lysine residues, quantifying polyubiquitin chain topologies, as well as profiling deubiquitinating enzymes based on the activity. Moreover, proteomic approaches have been developed for probing the interactome of proteasome and for identifying proteins with ubiquitin-binding domains. Similar strategies have been applied on the studies of the modification by ubiquitin-like proteins as well. These strategies are discussed with respect to their advantages, limitations and potential improvements. While the utilization of current methodologies has rapidly expanded the scope of protein modification by the ubiquitin family, a more active role is anticipated in the functional studies with the emergence of quantitative mass spectrometry.     

Functional proteomics in histone research and epigenetics

Post-translational modifications of histones comprise an important part of epigenetic gene regulation. Mass spectrometry and immunochemical techniques are currently the methods of choice for identification and quantitation of known and novel histone modifications. While peptide-centric mass spectrometry is a well-established tool for identification and quantification of histone modifications, recent technological advances have allowed discrete modification patterns to be assessed on intact histones. Chromatin immunoprecipitation assays (ChIP and ChIP-on-chip) are currently gaining tremendous popularity and are used to explore gene-specific patterns of histone modifications on a genomic scale. In this review, we introduce the basic concepts and recent developments of mass spectrometry, as well as immunochemical techniques and their applications in the analysis of histone modifications.