An introduction to hybrid ion trap/time‐of‐flight mass spectrometry coupled with liquid chromatography applied to drug metabolism studies

Metabolism studies play an important role at various stages of drug discovery and development. Liquid chromatography combined with mass spectrometry (LC/MS) has become a most powerful and widely used analytical tool for identifying drug metabolites. The suitability of different types of mass spectrometers for metabolite profiling differs widely, and therefore, the data quality and reliability of the results also depend on which instrumentation is used. As one of the latest LC/MS instrumentation designs, hybrid ion trap/time‐of‐flight MS coupled with LC (LC‐IT‐TOF‐MS) has successfully integrated ease of operation, compatibility with LC flow rates and data‐dependent MSⁿ with high mass accuracy and mass resolving power. The MSⁿ and accurate mass capabilities are routinely utilized to rapidly confirm the identification of expected metabolites or to elucidate the structures of uncommon or unexpected metabolites. These features make the LC‐IT‐TOF‐MS a very powerful analytical tool for metabolite identification. This paper begins with a brief introduction to some basic principles and main properties of a hybrid IT‐TOF instrument. Then, a general workflow for metabolite profiling using LC‐IT‐TOF‐MS, starting from sample collection and preparation to final identification of the metabolite structures, is discussed in detail. The data extraction and mining techniques to find and confirm metabolites are discussed and illustrated with some examples. This paper is directed to readers with no prior experience with LC‐IT‐TOF‐MS and will provide a broad understanding of the development and utility of this instrument for drug metabolism studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Deconvolution of Molecular Weight Distributions Using Dynamic Flory‐Schulz Distributions

Summary: The deconvolution of molecular weight distributions (MWDs) may be useful for obtaining information about the polymerization kinetics and properties of catalytic systems. However, deconvolution techniques are normally based on steady‐state assumptions and very little has been reported about the use of non‐stationary approaches for the deconvolution of MWDs. In spite of this, polymerization reactions are often performed in batch or semi‐batch modes. For this reason, dynamic solutions are proposed here for simple kinetic models and are then used for deconvolution of actual MWD data. Deconvolution results obtained with dynamic models are compared to deconvolution results obtained with the standard stationary Flory‐Schulz distributions. For coordination polymerizations, results show that dynamic MWD models are able to describe experimental data with fewer catalytic sites, which indicates that the proper interpretation of the reaction dynamics may be of fundamental importance for kinetic characterization. On the other hand, reaction dynamics induced by modification of chain transfer agent concentration seem to play a minor role in the shape of the MWD in free‐radical polymerizations.

This Figure illustrates that MWDs obtained at unsteady conditions should not be deconvoluted with standard steady‐state Flory‐Schulz distributions.     

Extended characterization of a vacuum gas oil by offline LC‐high‐temperature comprehensive two‐dimensional gas chromatography

In a context of environmental preservation, purification and conversion of heavy petroleum cuts into high‐quality fuel becomes essential. The interest for the characterization of those very complex matrices becomes a trendy analytical challenge, when it comes to get molecular information for the optimization of industrial processes. Among new analytical techniques, high‐temperature 2‐D GC has recently proved its applicability to heavy petroleum matrices, but lacks in selectivity to separate all chemical groups. To gain resolution, heart cutting is demonstrated for LC separation of saturated, aromatic and polar compounds prior to high‐temperature 2‐D GC. Therefore, an extended global resolution was obtained, especially by a better distinction of saturated compounds. This includes iso‐paraffins and biomarker polynaphthenic structures, which are impossible to quantify with MS methods. This new way to analyze heavy petroleum fractions gives innovative opportunities for the construction of global weight distributions by carbon atoms number and by chemical families. This can right now be employed for quantitative analysis of heavy petroleum fractions and for studying conversion processes.     

Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes

Chemical cross-linking of proteins, an established method in protein chemistry, has gained renewed interest in combination with mass spectrometric analysis of the reaction products for elucidating low-resolution three-dimensional protein structures and interacting sequences in protein complexes. The identification of the large number of cross-linking sites from the complex mixtures generated by chemical cross-linking, however, remains a challenging task. This review describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the strategies employing intra- or intermolecular chemical cross-linking and mass spectrometry. The various approaches described in the literature to facilitate detection of cross-linking products and also computer software for data analysis are reviewed. Cross-linking techniques combined with mass spectrometry and bioinformatic methods have the potential to provide the basis for an efficient structural characterization of proteins and protein complexes.     

Trace analysis of glyclazide in human plasma at microscale level by mass spectrometry

Green (reagents and organic solvents saving) analytical chemistry is a new strategy for pharmaceutical analysis. The principles of this idea include primary elimination or at least reduction of the amounts of organic reagents and solvents. In this study, we have provided two simple methods for the analysis of clinical drugs in human plasma. One is the capillary LC (Cap LC) connected to MS–MS, the other is the matrix‐assisted laser desorption ionization (MALDI) connected to TOF MS. Sulfonylurea drugs are usually used in diabetes mellitus patients. Diabetes is a syndrome of disordered metabolism resulting in abnormally high blood sugar levels (hyperglycemia). These microscale methods were successfully applied for the monitoring of drug levels in human plasma using gliclazide (a second‐generation sulfonylurea) as the test platform. The sensitivity of these methods is sufficient for detecting the gliclazide within a therapeutic range. All the analytical procedures (including human plasma, sample preparation, and flow rate of the analytical system) were at microscale level. These two methods would lower the consumption of organic solvents further safeguarding our environment.     

ESI,APCI, and MALDI tandem mass spectrometry of poly(methyl acrylate)s: A comparison study for the structural characterization of polymers synthesized via CRP techniques and the software application to analyze MS/MS data

Research in polymer science and engineering is moving from classical methodologies to advanced analytical strategies in which mass spectrometry (MS)‐based techniques play a crucial role. The molecular complexity of polymers requires new characterization tools and approaches to elucidate the detailed structural information. In this contribution, a comparison study of poly(methyl acrylate)s (PMA) using different tandem mass spectrometry techniques (ESI, APCI, and MALDI MS/MS) is reported to provide insights into the macromolecular structure with the aid of a special MS/MS data interpretation software. Collision‐induced dissociation (CID) was utilized to examine the fragmentation pathways of PMAs synthesized via various controlled radical polymerization techniques. All three mass spectrometry techniques are used to analyze structural details of PMAs and the labile end‐groups are determined based on the fragmentation behavior in CID. Fragmentation products were identified which are characteristics for the cleavage between the polymer chain and the end‐group. The application of a tailor‐made software is shown to analyze complex MS/MS data, and it is proven that this kind of software will be helpful for polymer scientists to identify fragmentation products obtained by tandem mass spectrometry similar to the fields of proteomics, metabolomics, genomics, and glycomics. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Cutting‐edge mass spectrometry characterization of originator,biosimilar and biobetter antibodies

The approval process for antibody biosimilars relies primarily on comprehensive analytical data to establish comparability and high similarity with the originator. Mass spectrometry (MS) in combination with liquid chromatography (LC) and electrophoretic methods are the corner stone for comparability and biosimilarity evaluation. In this special feature we report head‐to‐head comparison of trastuzumab and cetuximab with corresponding biosimilar and biobetter candidates based on cutting‐edge mass spectrometry techniques such as native MS and ion‐mobility MS at different levels (top, middle and bottom). In addition, we discuss the advantages and the limitations of sample preparation and enzymatic digestion, middle‐up and ‐down strategies and the use of hydrogen/deuterium exchange followed by MS (HDX‐MS). Last but not least, emerging separation methods combined to MS such as capillary zone electrophoresis‐tandem MS (CESI‐MS/MS), electron transfer dissociation (ETD), top down‐sequencing (TDS) and high‐resolution MS (HR‐MS) that complete the panel of state‐of‐the‐art MS‐based options for comparability and biosimilarity evaluation are presented. Copyright © 2015 John Wiley & Sons, Ltd.     

Protein dynamics revealed by hydrogen/deuterium exchange mass spectrometry: Correlation between experiments and simulation

Hydrogen deuterium exchange mass spectrometry (HDX‐MS) is a powerful technique for studying protein dynamics, which is an important factor governing protein functions. However, the process of hydrogen/deuterium exchange (HDX) of proteins is highly complex and the underlying mechanism has not yet been fully elucidated. Meanwhile, molecular dynamics (MD) simulation is a computational technique that can be used to elucidate HDX behaviour on proteins and facilitate interpretation of HDX‐MS data. This article aims to summarize the current understandings on the mechanism of HDX and its correlation with MD simulation, to discuss the recent developments in the techniques of HDX‐MS and MD simulation and to extend the perspectives of these two techniques in protein dynamics study.     

Recent advances in mass spectrometry analysis of low molecular weight heparins

Low molecular weight heparins (LMWHs) are the most widely used anticoagulant drugs produced by chemical or enzymatic modification of parent heparin polysaccharides. The present article reviews recent advances in orthogonal and complementary mass spectrometry (MS) methodologies towards complete elucidation of natural and modified structures in LMWHs that possibly affect the drug quality, safety and efficacy.     

Make them Blink: Probes for Super‐Resolution Microscopy

In recent years, a number of approaches have emerged that enable far‐field fluorescence imaging beyond the diffraction limit of light, namely super‐resolution microscopy. These techniques are beginning to profoundly alter our abilities to look at biological structures and dynamics and are bound to spread into conventional biological laboratories. Nowadays these approaches can be divided into two categories, one based on targeted switching and readout, and the other based on stochastic switching and readout of the fluorescence information. The main prerequisite for a successful implementation of both categories is the ability to prepare the fluorescent emitters in two distinct states, a bright and a dark state. Herein, we provide an overview of recent developments in super‐resolution microscopy techniques and outline the special requirements for the fluorescent probes used. In combination with the advances in understanding the photophysics and photochemistry of single fluorophores, we demonstrate how essentially any single‐molecule compatible fluorophore can be used for super‐resolution microscopy. We present examples for super‐resolution microscopy with standard organic fluorophores, discuss factors that influence resolution and present approaches for calibration samples for super‐resolution microscopes including AFM‐based single‐molecule assembly and DNA origami.     

Recent Advances in Analytical Methodology for in vivo Electrochemistry in Mammals

Electrochemistry is one of the most advanced techniques for monitoring neurochemical activities in the living brain because electrochemical approaches bear the advantageous features of high spatial and temporal resolutions, which facilitate its tremendous potential in investigating the highly spatially heterogeneous brain system and the fast dynamics of neurochemical activities. On the other hand, since brain is the most complicated organ in the sense of its numerous kinds of neurochemical species, high selectivity is always required for any analytical methods that approach the brain. In this review, we will discuss various electrochemical methodologies to achieve selective detection of neurochemicals in mammalian brain and the strategies developed mainly by our group towards selective monitoring of both electrochemically active and inactive neurochemicals. At the end, we will discuss possible solutions towards brain mapping of neurochemical species and combination of neurochemical detection strategy with electrophysiology as the direction of future development of electroanalysis in living brain.     

Evaluation of peptide fractionation strategies used in proteome analysis

Peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few comparisons of the relative separation efficiencies of 2‐D methodologies using complex biological samples have appeared, a systematic evaluation was conducted in this study. Four different fractionation methods, namely strong‐cation exchange, hydrophilic interaction chromatography, alkaline‐RP and solution isoelectric focusing, which can be used prior to LC‐MS/MS analysis, were compared. Strong‐cation exchange × RPLC was used after desalting the sample; significantly more proteins were identified, compared with the nondesalted sample (1990 and 1375). We also found that the use of a combination of analytical methods resulted in a dramatic increase in the number of unique peptides that could be identified, compared with only a small increase in protein levels. The increased number of distinct peptides that can be identified is especially beneficial, not only for unequivocally identifying proteins but also for proteomic studies involving posttranslational modifications and peptide‐based quantification approaches using stable isotope labeling. The identification and quantification of more peptides per protein provide valuable information that improves both the quantification of, and confidence of protein identification.     

New analytical strategies in studying drug metabolism

Identification and elucidation of the structures of metabolites play major roles in drug discovery and in the development of pharmaceutical compounds. These studies are also important in toxicology or doping control with either pharmaceuticals or illicit drugs. This review focuses on: new analytical strategies used to identify potential metabolites in biological matrices with and without radiolabeled drugs; use of software for metabolite profiling; interpretation of product spectra; profiling of reactive metabolites; development of new approaches for generation of metabolites; and detection of metabolites with increased sensitivity and simplicity. Most of the new strategies involve mass spectrometry (MS) combined with liquid chromatography (LC).     

Mass‐spectrometry‐based lipidomics

Lipids, which have a core function in energy storage, signalling and biofilm structures, play important roles in a variety of cellular processes because of the great diversity of their structural and physiochemical properties. Lipidomics is the large‐scale profiling and quantification of biogenic lipid molecules, the comprehensive study of their pathways and the interpretation of their physiological significance based on analytical chemistry and statistical analysis. Lipidomics will not only provide insight into the physiological functions of lipid molecules but will also provide an approach to discovering important biomarkers for diagnosis or treatment of human diseases. Mass‐spectrometry‐based analytical techniques are currently the most widely used and most effective tools for lipid profiling and quantification. In this review, the field of mass‐spectrometry‐based lipidomics was discussed. Recent progress in all essential steps in lipidomics was carefully discussed in this review, including lipid extraction strategies, separation techniques and mass‐spectrometry‐based analytical and quantitative methods in lipidomics. We also focused on novel resolution strategies for difficult problems in determining C=C bond positions in lipidomics. Finally, new technologies that were developed in recent years including single‐cell lipidomics, flux‐based lipidomics and multiomics technologies were also reviewed.     

Collision‐induced dissociative chemical cross‐linking reagent for protein structure characterization: applied Edman chemistry in the gas phase

Chemical cross‐linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross‐linked products presents an alternative approach to assess low‐resolution protein structures and to gain insight into protein interfaces. In this contribution, we report the design of an innovative cross‐linker based on Edman degradation chemistry, which leads to the formation of indicative mass shifted fragment ions and constant neutral losses (CNLs) in electrospray ionization (ESI)‐tandem‐mass spectrometry (MS/MS) product ion mass spectra, allowing an unambiguous identification of cross‐linked peptides. Moreover, the characteristic neutral loss reactions facilitate automated analysis by multiple reaction monitoring suited for high throughput studies with good sensitivity and selectivity. The functioning of the novel cross‐linker relies on the presence of a highly nucleophilic sulfur in a thiourea moiety, safeguarding for effective intramolecular attack leading to predictive and preferred cleavage of a glycyl‐prolyl amide bond. Our innovative analytical concept and the versatile applicability of the collision‐induced dissociative chemical cross‐linking reagent are exemplified for substance P, luteinizing hormone releasing hormone LHRH and lysozyme. The novel cross‐linker is expected to have a broad range of applications for probing protein tertiary structures and for investigating protein–protein interactions. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolite analysis in Curcuma domestica using various GC‐MS and LC‐MS separation and detection techniques

The metabolic profile of polar (methanol) and non‐polar (hexane) extracts of Curcuma domestica, a widely used medicinal plant, was established using various different analytical techniques, including GC‐FID, GC‐MS, HR‐GC‐MS and analytical HPLC‐ESI‐MS/MS by means of LTQ‐Orbitrap technology. The major non‐volatile curcuminoids curcumin, demethoxycurcumin and bisdemethoxycurcumin were identified when their chromatographic and precursor ion masses were compared with those of authentic standard compounds. In this paper we describe for the first time a GC/MS‐based method for metabolic profiling of the hydrophilic extract. We also identified 61 polar metabolites as TMS derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

Heart‐cut nano‐LC–CZE–MS for the characterization of proteins on the intact level

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed‐phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well‐suited geometries/dimensions. Here, a heart‐cut nano‐LC–CZE–MS setup was developed utilizing for the first time a mechanical 4‐port valve as LC–CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart‐cut transfer of individual LC peaks and subsequent CZE–MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower μg/mL range were determined, which are considerably lower compared to traditional CZE–MS. In addition, this study represents the first application of an LC–CE–MS system for intact protein analysis. The nano‐LC–CZE–MS system is expected to be applicable to various other analytical challenges.     

Structural determination of glycopeptidolipids of Mycobacterium smegmatis by high‐resolution multiple‐stage linear ion‐trap mass spectrometry with electrospray ionization

Glycopeptidolipids (GPLs) are abundant in the cell walls of different species of mycobacteria and consist of tripeptide‐amino‐alcohol core of D‐Phe‐D‐allo‐Thr‐D‐Ala‐L‐alaninol linked to 3‐hydroxy or 3‐methoxy C_26–34 fatty acyl chain at the N‐terminal of D‐Phe via amide linkage, and a 6‐deoxytalose (6‐dTal) and an O‐methyl rhamnose residues, respectively, attach to D‐allo‐Thr and the terminal L‐alaninol. They are important cell‐surface antigens that are implicated in the pathogenesis of opportunistic mycobacteria belonging to the Mycobacterium avium complex. In this contribution, we described multiple‐stage linear ion trap in conjunction with high‐resolution mass spectrometry towards structural characterization of complex GPLs as [M + Na]⁺ ions isolated from Mycobacterium smegmatis, a fast‐growing and non‐pathogenic mycobacterial species. Following resonance excitation in an ion trap, MSⁿ spectra of the [M + Na]⁺ ions of GPLs contained mainly b and y series ions that readily determine the peptide sequence. Fragment ions from MSⁿ also afford locating the 6‐dTal and O‐methyl rhamnose residues linked to the D‐allo‐Thr and terminal L‐alaninol of the peptide core, respectively, as well as recognizing the modifications of the glycosides, including their acetylation and methylation states and the presence of succinyl group. The GPL families consisting of 3‐hydroxy fatty acyl and of 3‐methoxy fatty acyl substituents are readily distinguishable. The MS profiles of the GPLs from cells are dependant on the conditions they were grown, and several isobaric isomers were identified for many of the molecular species. These multiple‐stage mass spectrometric approaches give detailed structures of GPL in complex mixtures of which the isomeric structures are difficult to define using other analytical methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Direct In Situ Investigation of Milling Reactions Using Combined X‐ray Diffraction and Raman Spectroscopy

The combination of two analytical methods including time‐resolved in situ X‐ray diffraction (XRD) and Raman spectroscopy provides a new opportunity for a detailed analysis of the key mechanisms of milling reactions. To prove the general applicability of our setup, we investigated the mechanochemical synthesis of four archetypical model compounds, ranging from 3D frameworks through layered structures to organic molecular compounds. The reaction mechanism for each model compound could be elucidated. The results clearly show the unique advantage of the combination of XRD and Raman spectroscopy because of the different information content and dynamic range of both individual methods. The specific combination allows to study milling processes comprehensively on the level of the molecular and crystalline structures and thus obtaining reliable data for mechanistic studies.