Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins

In the last 15 years substantial advances have been made to place isotope labels in native and glycosylated proteins for NMR studies and structure determination. Key developments include segmental isotope labeling using Native Chemical Ligation, Expressed Protein Ligation and Protein Trans-Splicing. These advances are pushing the size limit of NMR spectroscopy further making larger proteins accessible for this technique. It is just emerging that segmental isotope labeling can be used to define inter-domain interactions in NMR structure determination. Labeling of post-translational modified proteins like glycoproteins remains difficult but some promising developments were recently achieved. Key achievements are segmental and site-specific labeling schemes that improve resonance assignment and structure determination of the glycan moiety. We adjusted the focus of this perspective article to concentrate on the NMR applications based on recent developments rather than on labeling methods themselves to illustrate the considerable potential for biomolecular NMR.     

Application of Mass Spectrometry in Proteomics

Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.     

Interpretation of chemical shifts and coupling constants in macromolecules

Recent developments in NMR spectroscopy, along with advances in computational techniques, have produced new approaches to the interpretation of chemical shifts and spin-spin coupling constants in biomolecules. Quantum chemical studies of useful accuracy are now becoming more routine and are increasingly being used in conjunction with experimental studies to map out expected structural patterns for peptides and oligonucleotides. Topics of recent special interest include spin couplings across hydrogen bonds and patterns of chemical shift anisotropies, in both diamagnetic and paramagnetic proteins.     

Developments in solution-state NMR yield broader and deeper views of the dynamic ensembles of nucleic acids

Nucleic acids do not fold into a single conformation, and dynamic ensembles are needed to describe their propensities to cycle between different conformations when performing cellular functions. We review recent advances in solution-state nuclear magnetic resonance (NMR) methods and their integration with computational techniques that are improving the ability to probe the dynamic ensembles of DNA and RNA. These include computational approaches for predicting chemical shifts from structure and generating conformational libraries from sequence, measurements of exact nuclear Overhauser effects, development of new probes to study chemical exchange using relaxation dispersion, faster and more sensitive real-time NMR techniques, and new NMR approaches to tackle large nucleic acid assemblies. We discuss how these advances are leading to new mechanistic insights into gene expression and regulation.     

Improving human forensics through advances in genetics, genomics and molecular biology

Forensic DNA profiling currently allows the identification of persons already known to investigating authorities. Recent advances have produced new types of genetic markers with the potential to overcome some important limitations of current DNA profiling methods. Moreover, other developments are enabling completely new kinds of forensically relevant information to be extracted from biological samples. These include new molecular approaches for finding individuals previously unknown to investigators, and new molecular methods to support links between forensic sample donors and criminal acts. Such advances in genetics, genomics and molecular biology are likely to improve human forensic case work in the near future.     

Using NMR for ligand discovery and optimization

Several recent technology-driven advances in the area of NMR have rekindled an interest in the application of the technology to problems in drug discovery and development. A unique aspect of NMR is that it has applicability in broadly different areas of the drug discovery and optimization processes. NMR techniques for screening aimed at the discovery of novel ligands or low molecular weight structures for fragment-based build up procedures are being applied commonly in the industry. Application of NMR in structure-guided drug design and metabonomics are also becoming routine. We present an overview of some of the most recent NMR developments in these areas.     

Structural elucidation of low abundant metabolites in complex sample matrices

Identification of metabolites is a major challenge in biological studies and relies in principle on mass spectrometry (MS) and nuclear magnetic resonance (NMR) methods. The increased sensitivity and stability of both NMR and MS systems have made dereplication of complex biological samples feasible. Metabolic databases can be of help in the identification process. Nonetheless, there is still a lack of adequate spectral databases that contain high quality spectra, but new developments in this area will assist in the (semi-)automated identification process in the near future. Here, we discuss new developments for the structural elucidation of low abundant metabolites present in complex sample matrices. We describe how a recently developed combination of high resolution MS multistage fragmentation (MS ⁿ ) and high resolution one dimensional (1D)-proton (¹H)-NMR of liquid chromatography coupled to solid phase extraction (LC–SPE) purified metabolites can circumvent the need for isolating extensive amounts of the compounds of interest to elucidate their structures. The LC–MS–SPE–NMR hardware configuration in conjunction with high quality databases facilitates complete structural elucidation of metabolites even at sub-microgram levels of compound in crude extracts. However, progress is still required to optimally exploit the power of an integrated MS and NMR approach. Especially, there is a need to improve and expand both MS ⁿ and NMR spectral databases. Adequate and user-friendly software is required to assist in candidate selection based on the comparison of acquired MS and NMR spectral information with reference data. It is foreseen that these focal points will contribute to a better transfer and exploitation of structural information gained from diverse analytical platforms.     

Maximizing content across scales: Moving multimodal microscopy and mesoscopy toward molecular imaging

Molecular imaging aims to depict the molecules in living patients. However, because this aim is still far beyond reach, patchworks of different solutions need to be used to tackle this overarching goal. From the vast toolbox of imaging techniques, we focus on those recent advances in optical microscopy that image molecules and cells at the submicron to centimeter scale. Mesoscopic imaging covers the “imaging gap” between techniques such as confocal microscopy and magnetic resonance imagingthat image entire live samples but with limited resolution. Microscopy focuses on the cellular level; mesoscopy visualizes the organization of molecules and cells into tissues and organs. The correlation between these techniques allows us to combine disciplines ranging from whole body imaging to basic research of model systems. We review current developments focused on improving microscopic and mesoscopic imaging technologies and on hardware and software that push the current sensitivity and resolution boundaries.     

Flow NMR applications in combinatorial chemistry

Flow NMR techniques are now well accepted and widely used in many areas of drug discovery. Although natural-product-, rational-drug-design-, and NMR-screening-programs have begun to use flow NMR more routinely, flow NMR has not yet gained widespread acceptance in combinatorial chemistry, even though it has been shown to be a potentially useful tool. Recent developments in DI-NMR, FIA-NMR, and LC-NMR will help flow NMR eventually gain a wider acceptance within combinatorial chemistry. These developments include LC-NMR-MS instrumentation, flow probe improvements, new pulse sequences, improved automation of NMR data analysis, and the application of flow NMR to related fields in drug discovery.     

Twenty years of research into medicinal plants: Results and perspectives

Over the years 1981 to 2001 there has been a rapid evolution of research into medicinal plants. The major improvement has been the introduction of simple and predictive bioassays for bioactivity-guided isolation. Radical developments in separation methods have also taken place. Another important addition has been the development of hyphenated techniques involving HPLC: LC/UV, LC/MS, LC/MSⁿ and LC/NMR. These are indispensable nowadays for the early detection and identification of new compounds in crude plant extracts. Hyphenated techniques allow an efficient targeted isolation approach for the discovery of new lead compounds. Other areas of increasing importance include the investigation of toxic constituents of plants and phytomedicines, and the effects of genetic modifications on plant secondary metabolites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microscale NMR

NMR spectroscopy is increasingly being used to characterize microliter and smaller-volume samples. Substances at picomole levels have been identified using NMR spectrometers equipped with microcoil-based probes. NMR probes that incorporate multiple sample chambers enable higher-throughput NMR experiments. Hyphenation of capillary-scale separations and microcoil NMR has also decreased analysis time of mixtures. For example, capillary isotachophoresis/NMR allows the highest mass sensitivity nanoliter-volume flow cells to be used with low microliter volume samples because isotachophoresis concentrates the microliter volume sample into the nanoliter volume NMR detection probe. In addition, the diagnostic capabilities of NMR spectroscopy allow the physico-chemical aspects of a capillary separation process to be characterized on-line. Because of such advances, the application of NMR to smaller samples continues to grow.     

Developments in multiple headspace extraction

This paper reviews new developments in multiple headspace extraction (MHE), especially its combination with two miniaturized extraction techniques, solid-phase microextraction (SPME) and single-drop microextraction (SDME). The combination of the techniques broadens the applicability of SPME and SDME to quantitative determination of analytes in complex liquid and solid matrixes. These new methods offer several advantages over traditional liquid-solid, liquid-liquid and headspace extraction techniques. The potential applications include extraction of volatiles and semivolatiles from environmental and physiological samples and from different polymer products such as medical and biomedical materials, food packaging and building materials. The theoretical principals of the techniques are also briefly reviewed.     

Developments in multiple headspace extraction

This paper reviews new developments in multiple headspace extraction (MHE), especially its combination with two miniaturized extraction techniques, solid-phase microextraction (SPME) and single-drop microextraction (SDME). The combination of the techniques broadens the applicability of SPME and SDME to quantitative determination of analytes in complex liquid and solid matrixes. These new methods offer several advantages over traditional liquid–solid, liquid–liquid and headspace extraction techniques. The potential applications include extraction of volatiles and semivolatiles from environmental and physiological samples and from different polymer products such as medical and biomedical materials, food packaging and building materials. The theoretical principals of the techniques are also briefly reviewed.     

Interventional neuroradiology.

A wide variety of diseases affecting the central nervous system and head and neck can be treated using interventional neuroradiologic techniques. These new treatments have depended on advances in radiologic imaging, catheter technology, and the development of new embolic agents. These procedures may be an adjunct to other therapy, palliative or curative. Diseases for which interventional neuroradiologic techniques have been major advances in treatment include cerebral aneurysms, vasospasm after subarachnoid hemorrhage, cerebral arteriovenous malformations, dural arteriovenous fistulas, dural sinus thrombosis, atherosclerosis, scalp arteriovenous fistulas, carotid-cavernous fistulas, and stroke. This field is rapidly evolving as advances are made in catheter technology and new embolic agents are developed.     

Molecular biology and gene transfer in atherosclerosis in the stenting era

Atherosclerosis is the major cause of death in the developed world. Understanding the pathogenesis of atherosclerosis has been a major challenge to cardiovascular research over the past several decades. During this period a number of advances in various scientific disciplines has increased our understanding of this disease. These include improved understanding of the structural and functional components of normal vessel wall and more recently the use of cell biology and molecular biology techniques to elucidate the pathogenesis of atherosclerosis. None of these advances has been more dramatic nor has potentially more far reaching consequences as the application of molecular biology and gene technology to the practice of cardiovascular medicine. These developments have already opened new and exciting areas of vascular research and may in the future provide for earlier identification of genetic predisposition to atherosclerosis, strategic planning of preventive therapy and more tailored pharmacologic approaches for established disease.     

Hyperpolarized NMR metabolomics

Hyperpolarized NMR is a promising approach to address the sensitivity limits of conventional NMR metabolomics approaches, which currently fails to detect minute metabolite concentrations in biological samples. This review describes how tremendous signal enhancement offered by dissolution-dynamic nuclear polarization and parahydrogen-based techniques can be fully exploited for molecular omics sciences. Recent developments, including the combination of hyperpolarization techniques with fast multi-dimensional NMR implementation and quantitative workflows are described, and a comprehensive comparison of existing hyperpolarization techniques is proposed. High-throughput, sensitivity, resolution and other relevant challenges that should be tackled for a general application of hyperpolarized NMR in metabolomics are discussed.     

X-ray crystallography of chemical compounds

AimsAccurate knowledge of molecular structure is a prerequisite for rational drug design. This review examines the role of X-ray crystallography in providing the required structural information and advances in the field of X-ray crystallography that enhance or expand its role.Main methodsX-ray crystallography of new drugs candidates and intermediates can provide valuable information of new syntheses and parameters for quantitative structure activity relationships (QSAR).Key findingsCrystallographic studies play a vital role in many disciplines including materials science, chemistry, pharmacology, and molecular biology. X-ray crystallography is the most comprehensive technique available to determine molecular structure. A requirement for the high accuracy of crystallographic structures is that a ‘good crystal’ must be found, and this is often the rate-limiting step. In the past three decades developments in detectors, increases in computer power, and powerful graphics capabilities have contributed to a dramatic increase in the number of materials characterized by X-ray crystallography. More recently the advent of high-throughput crystallization techniques has enhanced our ability to produce that one good crystal required for crystallographic analysis.SignificanceContinuing advances in all phases of a crystallographic study have expanded the ranges of samples which can be analyzes by X-ray crystallography to include larger molecules, smaller or weakly diffracting crystals, and twinned crystals.     

Genome comparisons and analysis

As we enter the post-genomic era, with the accelerating availability of complete genome sequences, new theoretical approaches and new experimental techniques, our ability to dissect cellular processes at the molecular level continues to expand. Recent advances include the application of RNA interference methods to characterize loss-of-function phenotype genes in higher eukaryotes, comparative analysis of the human and mouse genome sequences, and methods for reconciling contradictory phylogenetic reconstructions. New developments feed into the increasingly rich content of databases such as the COG database. The next phase of research will be increasingly dominated by efforts to integrate the deluge of data into our understanding of biological systems.     

Resonance assignment strategies for the analysis of nmr spectra of proteins

Determination of the high resolution solution structure of a protein using nuclear magnetic resonance (NMR) spectroscopy requires that resonances observed in the NMR spectra be unequivocally assigned to individual nuclei of the protein. With the advent of modern, two-dimensional NMR techniques arose methodologies for assigning the¹H resonances based on 2D, homonuclear¹H NMR experiments. These include the sequential assignment strategy and the main chain directed strategy. These basic strategies have been extended to include newer 3D homonuclear experiments and 2D and 3D heteronuclear resolved and edited methods. Most recently a novel, conceptually new approach to the problem has been introduced that relies on heteronuclear, multidimensional so-called triple resonance experiments for both backbone and sidechain resonance assignments in proteins. This article reviews the evolution of strategies for the assignment of resonances of proteins.