Metrology for metalloproteins—where are we now, where are we heading?

The use of the amount of certain proteins in biological samples as markers for distinguishing between a healthy and a diseased state has become increasingly important in clinical diagnosis. As about 30 % of all proteins contain metals in one form or another, either as a cofactor or covalently bound as part of the protein, some of these proteins are regularly analyzed in clinical laboratories. With the increasing number of measurements of those proteins performed all over the world, the necessity of obtaining reliable and comparable results is becoming a focal point for scientists and politicians. Directives such as the EC directive covering in vitro diagnostic medical devices (Directive 98/79/EC) and standards such as EN ISO 17511:2003 demand the traceability of the results obtained for analytes in samples of human origin. However, no reference measurement procedures with results traceable to the SI exist for many metalloproteins. In this article, the situation for a few important metalloproteins, such as hemoglobin, transferrin, superoxide dismutase, ceruloplasmin, and C-reactive protein, for which specific efforts have been made in recent years to achieve comparable and traceable results worldwide, is discussed. These proteins also serve as examples of the difficulties scientists face when they wish to quantify proteins and the pitfalls they should avoid to achieve reliable results.

A decade of yeast surface display technology: where are we now?

Yeast surface display has become an increasingly popular tool for protein engineering and library screening applications. Recent advances have greatly expanded the capability of yeast surface display, and are highlighted by cell-based selections, epitope mapping, cDNA library screening, and cell adhesion engineering. In this review, we discuss the state-of-the-art yeast display methodologies and the rapidly expanding set of applications afforded by this technology.     

An insight into iTRAQ: where do we stand now?

The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ's attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.     

Improving the intrinsic activity of electrocatalysts for sustainable energy conversion: where are we and where can we go?

As we are in the midst of a climate crisis, there is an urgent need to transition to the sustainable production of fuels and chemicals. A promising strategy towards this transition is to use renewable energy for the electrochemical conversion of abundant molecules present in the earth''s atmosphere such as H₂O, O₂, N₂ and CO₂, to synthetic fuels and chemicals. A cornerstone to this strategy is the development of earth abundant electrocatalysts with high intrinsic activity towards the desired products. In this perspective, we discuss the importance and challenges involved in the estimation of intrinsic activity both from the experimental and theoretical front. Through a thorough analysis of published data, we find that only modest improvements in intrinsic activity of electrocatalysts have been achieved in the past two decades which necessitates the need for a paradigm shift in electrocatalyst design. To this end, we highlight opportunities offered by tuning three components of the electrochemical environment: cations, buffering anions and the electrolyte pH. These components can significantly alter catalytic activity as demonstrated using several examples, and bring us a step closer towards complete system level optimization of electrochemical routes to sustainable energy conversion.

We evaluate the improvements over the past two decades in intrinsic activity of electrocatalysts for sustainable energy conversion, and highlight opportunities from tuning the electrolyte.     

Electrochemistry: quo vadis or where should we head to?

Journal of Solid State Electrochemistry -     

The impact of flow injection on modern chemical analysis: has it fulfilled our expectations? And where are we going?

Presenting a condensation of the opening lecture of the 12th ICFIA conference, this communication presents a view of the impact that flow injection analysis (FIA) has had on modern analytical chemistry, evaluated both within the academic community and outside it, i.e. in “industry”. The ensuing developments of FIA, encompassing sequential injection analysis (SIA) and bead injection lab-on-valve (BI-LOV), are described and their individual features discussed. Finally, some recent results of the activities from the author’s own research group are briefly mentioned.     

Ion spectroscopy: Where did it come from; where is it now; and where is it going?

The ASMS conference on ion spectroscopy brought together at Asilomar on October 16–20, 2009 a large group of mass spectrometrists working in the area of ion spectroscopy. In this introduction to the field, we provide a brief history, its current state, and where it is going. Ion spectroscopy of intermediate size molecules began with photoelectron spectroscopy in the 1960s, while electronic spectroscopy of ions using the photodissociation “action spectroscopic” mode became active in the next decade. These approaches remained for many years the main source of information about ionization energies, electronic states, and electronic transitions of ions. In recent years, high-resolution laser techniques coupled with pulsed field ionization and sample cooling in molecular beams have provided high precision ionization energies and vibrational frequencies of small to intermediate sized molecules, including a number of radicals. More recently, optical parametric oscillator (OPO) IR lasers and free electron lasers have been developed and employed to record the IR spectra of molecular ions in either molecular beams or ion traps. These results, in combination with theoretical ab initio molecular orbital (MO) methods, are providing unprecedented structural and energetic information about gas-phase ions.     

What are we determining using gas chromatographic multiresidue methods: tralomethrin or deltamethrin?

The analytical behaviour of the relatively new pyrethroid insecticide tralomethrin has been evaluated by using gas chromatography (GC) with electron-capture and mass spectrometry (MS) detectors, and liquid chromatography (LC)-atmospheric pressure ionization mass spectrometry with electrospray interfacing. Under the GC conditions commonly used in pesticide residue analysis, it was found that tralomethrin is transformed into deltamethrin (in a reproducible way) in the injector port of the GC system. Results obtained in this work indicate that the GC multiresidue methodologies routinely applied in the analysis of pyrethroid pesticides in foods cannot distinguish between these two pesticides, and the chromatographic signal obtained at the retention time of deltamethrin/tralomethrin can be really quantified as either deltamethrin or tralomethrin, including when it is confirmed as deltamethrin by MS. Under the LC-MS conditions assessed in this work, deltamethrin and the two diasteroisomers of tralomethrin were well separated and identified.     

Current trends in lead discovery: are we looking for the appropriate properties?

The new drug discovery paradigm is based on high-throughput technologies, both with respect to synthesis and screening. The progression HTS hits lead series candidate drug marketed drug appears to indicate that the probability of reaching launched status is one in a million. This has shifted the focus from good quality candidate drugs to good quality leads. We examined the current trends in lead discovery by comparing MW (molecular weight), LogP (octanol/water partition coefficient, estimated by Kowwin [17]) and LogSw (intrinsic water solubility, estimated by Wskowwin [18]) for the following categories: 62 leads and 75 drugs [11]; compounds in the development phase (I, II, III and launched), as indexed in MDDR; and compounds indexed in medicinal chemistry journals [ref. 20], categorized according to their biological activity. Comparing the distribution of the above properties, the 62 lead structures show the lowest median with respect to MW (smaller) and LogP (less hydrophobic), and the highest median with respect to LogSw (more soluble). By contrast, over 50% of the medicinal chemistry compounds with activities above 1 nanomolar have MW > 425, LogP > 4.25 and LogSw < -4.75, indicating that the reported active compounds are larger, more hydrophobic and less soluble when compared to time-tested quality leads. In the MDDR set, a progressive constraint to reduce MW and LogP, and to increase LogSw, can be observed when examining trends in the developmental sequence: phase I, II, III and launched drugs. These trends indicate that other properties besides binding affinity, e.g., solubility and hydrophobicity, need to be considered when choosing the appropriate leads.     

A universal approach for continuum solvent pK a calculations: are we there yet?

This paper reviews several pK _a calculation strategies that are commonly used in aqueous acidity predictions. Among those investigated were the direct or absolute method, the proton exchange scheme, and the hybrid cluster–continuum (Pliego and Riveros) and implicit–explicit (Kelly, Cramer and Truhlar) models. Additionally, these protocols are applied in the pK _a calculation of 55 neutral organic and inorganic acids in conjunction with various solvent models, including the CPCM-UAKS/UAHF, IPCM, SM6 and COSMO-RS, with a view to identifying a universal approach for accurate pK _a predictions. The results indicate that the direct method is unsuitable for general pK _a calculations, although moderately accurate results (MAD <3 units) are possible for certain classes of acids, depending on the choice of solvent model. The proton exchange scheme generally delivers good results (MAD <2 units), with CPCM-UAKS giving the best performance. Furthermore, the sensitivity of this approach to the choice of reference acid can be substantially lessened if the solvation energies for ionic species are calculated via the IPCM cluster–continuum approach. Reference-independent hybrid approaches that include explicit water molecules can potentially give reasonably accurate values (MAD generally ~2 units) depending on the solvent model and the number of explicit water molecules added.     

Polymer modeling: where has it been and where is it going?

The development of the area of polymer modeling often referred to as molecular modeling has been reviewed from its early beginnings to the present day. Key forces influencing the development include computational power, algorithmic advances and access to computational resources. The desire to apply modeling techniques to predict the properties of increasingly complex polymer-containing systems, taken in conjunction with a number of current limitations discussed in this brief review, is expected to define in part some essential future developments.     

Solid-phase microextraction. How far are we from clinical practice?

The current review briefly discusses the future development of solid-phase microextraction (SPME) and its potential application in the field of clinical medicine, including pharmacokinetic studies, therapeutic drug monitoring, biomarker discovery, and targeted and untargeted metabolomics. We also discuss aspects of automation and high-throughput analysis as major requirements of daily clinical practice. We give examples of clinically-validated applications of SPME and point out the regulatory restrictions limiting some in-vivo SPME studies. We briefly review the current state of progress in this extraction technique in the context of its future application in medical research and laboratory testing, including new directions (i.e. personalized medicine).     

Speciation and legislation – Where are we today and what do we need for tomorrow?

In international legislation concerning trace elements in food, in the environment or in occupational health most regulations are based on the total element contents, and are frequently given as maximum limits or guideline levels. In contrast, only few regulations pay attention to the molecular species in which the elements are bound. The international legislation concerning contaminants in food is presently being established in the Codex Alimentarius, which is an independent United Nations organisation under the joint FAO/WHO Food Standards Programme. Development of the Codex General Standard for Contaminants and Toxins in Food provides the framework for future international legislation on metals as contaminants in food. For certain food additives, which include some essential minerals, speciation is an integral part of the set of specification criteria, because only certain defined chemical compounds are permitted as sources of the essential element. The development of more species-specific analytical and toxicological data, and improved communication with legislators will be necessary before it will become possible to lay down species-specific regulations in all the cases where the specialised scientist will consider it reasonable.     

Long-term tracking of cells using inorganic nanoparticles as contrast agents: are we there yet?

The use of inorganic nanoparticles as probes to label and track cells in vivo is already a reality. While superparamagnetic nanoparticles have been the subject of clinical studies involving magnetic resonance imaging, quantum dots and gold nanoparticles are starting to be explored for similar goals in pre-clinical studies involving fluorescence and photoacoustic imaging. Although exciting results have been obtained from in vivo investigations, there appears to be a general lack of understanding on the effects of physicochemical properties on the labelling efficiency and toxicity of those nanoparticles, as well as on their stability in the intracellular microenvironment; essential requirements for using them as probes for cellular tracking. In this tutorial review, we look at what the current literature can teach us in respect to cell interactions with these nanoparticles, with the perspective of using them as probes for cell labelling. We also examine the findings obtained in pre-clinical studies that expose potential misinterpretation that can occur when using inorganic nanoparticles for in vivo imaging.     

PDLC shutters: where has this technology gone?

A Commentary on the paper ”Response times and voltages for PDLC shutters?, by Bao‐Gang Wu, John H. Erdman and J. William Doane. First published in Liquid Crystals, 5, 1453‐1465 (1989).     

Accuracy of enthalpy and entropy determination using the kinetic method: are we approaching a consensus?

There is an emerging consensus regarding the applicability of the kinetic method. All parties acknowledge that it is an approximate quantitative technique, capable of yielding not only enthalpy, but also entropy values. Opinions differ mainly on the accuracy of the results but it is agreed that the energy (effective temperature) dependence of kinetic method plots needs to be checked in all but the simplest of cases. When the 'apparent basicity' is found to depend on collision energy (and hence effective temperature), the extended kinetic method must be used. We have performed a large-scale modeling study, involving thousands of randomly selected molecular systems and a variety of experimental conditions, using exact calculations and realistic data sets. The results show that when the measured entropy difference between the two competing reaction channels is less than approximately 35 J mol(-1) K(-1), overall errors (standard deviations) of DeltaH(298) determined by the kinetic method are +/-5 kJ mol(-1); those of DeltaS(298) are +/-10 J mol(-1) K(-1). These include not only inherent errors of the kinetic method, but also errors in ion abundance measurement (5%) and inaccurate knowledge of reference compound thermochemistry (+/-2 kJ mol(-1), on average). We recommend, in general, that these errors be reported in kinetic method studies. When the measured entropy difference between the two competing fragmentation channels is large (>35 J mol(-1) K(-1)), it is likely to be significantly underestimated and errors of the kinetic method increase significantly.     

Carbohydrate-pi interactions: what are they worth?

Protein-carbohydrate interactions play an important role in many biologically important processes. The recognition is mediated by a number of noncovalent interactions, including an interaction between the alpha-face of the carbohydrate and the aromatic side chain of the protein. To elucidate this interaction, it has been studied in the context of a beta-hairpin in aqueous solution, in which the interaction can be investigated in the absence of other cooperative noncovalent interactions. In this beta-hairpin system, both the aromatic side chain and the carbohydrate were varied in an effort to gain greater insight into the driving force and magnitude of the carbohydrate-pi interaction. The magnitude of the interaction was found to vary from -0.5 to -0.8 kcal/mol, depending on the nature of the aromatic ring and the carbohydrate. Replacement of the aromatic ring with an aliphatic group resulted in a decrease in interaction energy to -0.1 kcal/mol, providing evidence for the contribution of CH-pi interactions to the driving force. These findings demonstrate the significance of carbohydrate-pi interactions within biological systems and also their utility as a molecular recognition element in designed systems.