A review of current applications of mass spectrometry for neuroproteomics in epilepsy.

The brain is unquestionably the most fascinating organ, and the hippocampus is crucial in memory storage and retrieval and plays an important role in stress response. In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation. Despite tremendous progress, current knowledge falls short of being able to explain its function. An emerging approach toward an improved understanding of the complex molecular mechanisms that underlie functions of the brain and hippocampus is neuroproteomics. Mass spectrometry has been widely used to analyze biological samples, and has evolved into an indispensable tool for proteomics research. In this review, we present a general overview of the application of mass spectrometry in proteomics, summarize neuroproteomics and systems biology-based discovery of protein biomarkers for epilepsy, discuss the methodology needed to explore the epileptic hippocampus proteome, and also focus on applications of ingenuity pathway analysis (IPA) in disease research. This neuroproteomics survey presents a framework for large-scale protein research in epilepsy that can be applied for immediate epileptic biomarker discovery and the far-reaching systems biology understanding of the protein regulatory networks. Ultimately, knowledge attained through neuroproteomics could lead to clinical diagnostics and therapeutics to lessen the burden of epilepsy on society.

ICP-MS-based strategies for protein quantification.

In the post-genomics era, proteomics has become a central branch in life sciences. An understanding of biological functions will not only rely on protein identification, but also on protein quantification in a living organism. Most of the existing methods for quantitative proteomics are based on isotope labeling combined with molecular mass spectrometry. Recently, a remarkable progress that utilizes inductively coupled plasma-mass spectrometry (ICP-MS) as an attractive complement to electrospray MS and MALDI MS for protein quantification, especially for absolute quantification, has been achieved. This review will selectively discuss the recent advances of ICP-MS-based technique, which will be expected to further mature and to become one of the key methods in quantitative proteomics.

Current trends in computational inference from mass spectrometry-based proteomics.

Mass spectrometry offers a high-throughput approach to quantifying the proteome associated with a biological sample and hence has become the primary approach of proteomic analyses. Computation is tightly coupled to this advanced technological platform as a required component of not only peptide and protein identification, but quantification and functional inference, such as protein modifications and interactions. Proteomics faces several key computational challenges such as identification of proteins and peptides from tandem mass spectra as well as their quantitation. In addition, the application of proteomics to systems biology requires understanding the functional proteome, including how the dynamics of the cell change in response to protein modifications and complex interactions between biomolecules. This review presents an overview of recently developed methods and their impact on these core computational challenges currently facing proteomics.

Site-specific protein modification: advances and applications.

Although chemical methods to modify proteins in a sequence-specific manner have yet to be developed, site-specific post-translational modification of proteins has recently emerged as a major focus in biological chemistry. Post-translational modification with functionalized substrate analogues opens up several unique avenues to induce selective reactivity into proteins in a sequence-specific manner, and can be applied to protein identification and manipulation in both in vitro and in vivo contexts. Further in vivo applications of this method will enable the imaging of cellular processes, avoiding nonspecific labeling and probe scattering, major complications observed in nonenzymatic methods. Additionally, new tools for in vitro protein modification have been developed that offer more versatile ways to study protein structure and function.

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.

The evolution of tools for protein phosphorylation site analysis: from discovery to clinical application.

The importance of the analysis of signaling pathways has been proven for many years by the elucidation of key signaling molecules. However, in most cases these pathways tend to represent a rather narrow view of the biological state under investigation. Clearly a more detailed understanding of the complexities of cross-talk between signaling pathways is required to further our knowledge of normal and disease processes. The tools that provide the framework for this increased understanding of biology, those that enable identification, characterization, and quantitation of sites of phosphorylation in proteins, have advanced over the past 25 years. This review will present a brief overview of the history of the tools used in phosphorylation analysis and the latest technologies that are being applied in this field, such as mass spectrometry (for broad-based discovery efforts) and flow cytometry (for translation to clinical applications).

Mass spectrometry: m/z 1983-2008.

While definitely not a new technology, mass spectrometry (MS) has seen incredible growth over the past 25 years. Mass spectrometry has rapidly evolved to the forefront of analytical techniques; its ability to analyze proteins is the major driving force in the field of proteomics. MS instrumentation has increased approximately 5-fold in sensitivity every three years. The level of performance that is achievable with MS today allows scientists to study proteins in ways that were inconceivable a quarter century ago. This review of the history of MS over the past 25 years is timely in that it encompasses two of the biggest developments, electrospray and matrix-assisted laser desorption/ionization (MALDI), which have enabled many of the uses of this technology today.

Is charge reduction in ESI really necessary?

The technique of charge reduction electrospray mass spectrometry (CREMS), which can reduce the charge state complexity produced in electrospray ionization (ESI), is discussed.

Porous silicon protein microarray technology and ultra-/superhydrophobic states for improved bioanalytical readout.

One attractive method for monitoring biomolecular interactions in a highly parallel fashion is the use of microarrays. Protein microarray technology is an emerging and promising tool for protein analysis, which ultimately may have a large impact in clinical diagnostics, drug discovery studies and basic protein research. This chapter is based upon several original papers presenting our effort in the development of new protein microarray chip technology. The work describes a novel 3D surface/platform for protein characterization based on porous silicon. The simple adjustment of pore morphology and geometry offers a convenient way to control wetting behavior of the microarray substrates. In this chapter, an interesting insight into the surface role in bioassays performance is made. The up-scaled fabrication of the novel porous chips is demonstrated and stability of the developed supports as well as the fluorescent bioassay reproducibility and data quality issues are addressed. We also describe the efforts made by our group to link protein microarrays to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), suggesting porous silicon as a convenient platform for fast on-surface protein digestion protocols linked to MS-readout. The fabrication of ultra- and superhydrophobic states on porous silicon is also described and the utilization of these water-repellent properties for a new microscaled approach to superhydrophobic MALDI-TOF MS target anchor chip is covered.

Microbial metabolomics: past, present and future methodologies.

Microbial metabolomics has received much attention in recent years mainly because it supports and complements a wide range of microbial research areas from new drug discovery efforts to metabolic engineering. Broadly, the term metabolomics refers to the comprehensive (qualitative and quantitative) analysis of the complete set of all low molecular weight metabolites present in and around growing cells at a given time during their growth or production cycle. This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites as well as quantification of these metabolites using enzyme based and or modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (LC-MS(n), GC-MS(n), CE-MS(n)), where n indicates the number of tandem mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR).

A dynamic look backward and forward.

The 2015 Gunther Laukien Prize recognized solution NMR studies of protein dynamics and thermodynamics. This Perspective surveys aspects of the development and application of NMR spin relaxation for investigations of protein flexibility and function over multiple time scales in solution. Methods highlighted include analysis of overall rotational diffusion, theoretical descriptions of R1ρ relaxation, and molecular dynamics simulations to interpret NMR spin relaxation. Applications are illustrated for the zinc-finger domain Xfin-31, the calcium-binding proteins calbindin D9k and calmodulin, and the bZip transcription factor of GCN4.

Proteomics--post-genomic cartography to understand gene function.

The completion of the genomic sequences of numerous organisms from human and mouse to Caenorhabditis elegans and many microorganisms, and the definition of their genes provides a database to interpret cellular protein-expression patterns and relate them to protein function. Proteomics technologies that are dependent on mass spectrometry and involve two-dimensional gel electrophoresis are providing the main window into the world of differential protein-expression analysis. In this article, the limitations and expectations of this research field are examined and the future of the analytical needs of proteomics is explored.

Proteomics: quantitative and physical mapping of cellular proteins.

Genome sequencing provides a wealth of information on predicted gene products (mostly proteins), but the majority of these have no known function. Two-dimensional gel electrophoresis and mass spectrometry have, coupled with searches in protein and EST databases, transformed the protein-identification process. The proteome is the expressed protein complement of a genome and proteomics is functional genomics at the protein level. Proteomics can be divided into expression proteomics, the study of global changes in protein expression, and cell-map proteomics, the systematic study of protein-protein interactions through the isolation of protein complexes.

Future directions of structural mass spectrometry using hydroxyl radical footprinting.

Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein-protein and protein-DNA interactions. Using synchrotron radiolysis, exposure of proteins to a 'white' X-ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time-resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium-dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time-resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)-based method can be utilized for quantification of oxidized species, improving the signal-to-noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis-driven structural mass spectrometry experiments.

How far can we go with structural mass spectrometry of protein complexes?

Physical interactions between proteins and the formation of stable complexes form the basis of most biological functions. Therefore, a critical step toward understanding the integrated workings of the cell is to determine the structure of protein complexes, and reveal how their structural organization dictates function. Studying the three-dimensional organization of protein assemblies, however, represents a major challenge for structural biologists, due to the large size of the complexes, their heterogeneous composition, their flexibility, and their asymmetric structure. In the last decade, mass spectrometry has proven to be a valuable tool for analyzing such noncovalent complexes. Here, I illustrate the breadth of structural information that can be obtained from this approach, and the steps taken to elucidate the stoichiometry, topology, packing, dynamics, and shape of protein complexes. In addition, I illustrate the challenges that lie ahead, and the future directions toward which the field might be heading.