Time-Resolved Mass Spectrometry: From Concept to Applications

Author Biographies

Pawel Lukasz Urban received his education from the University of Warsaw (MSc) and the University of York (PhD). His research training was supplemented with pre- and post-doctoral stays at the University of Alcala and ETH Zurich. He currently holds an academic position at the National Chiao Tung University. His research and teaching interests encompass biochemical analysis, development of instrumentation, and engineering smart biosystems.

Yu-Chie Chen received her education from the National Sun Yat-Sen University (MSc) and Montana State University (PhD). She is Professor of Chemistry at the National Chiao Tung University. Her research interests include biological mass spectrometry, nanomedicine, and nanotechnology. She is the co-inventor of several ionization techniques for mass spectrometry (SALDI, UASI, C-API, PI-ESI), which are useful in the monitoring of chemical reactions.

Yi-Sheng Wang received his education from the Feng Chia University (BEng) and the National Taiwan University (PhD). He is currently Associate Research Fellow in the Genomics Research Center of Academia Sinica. His research covers in-depth developments in mass spectrometry instrumentation, fundamental research on ionization chemistry, method development and applications of biological mass spectrometry.

Preface

Mass spectrometry has become an important part of teaching curricula in many university programs related to chemistry, biology, physics, and engineering. The field is highly interdisciplinary. This book takes the readers into the field of time-resolved mass spectrometry (TRMS) providing more detailed conceptual background and practical guidance than the specialized review papers published in the past few years. It is intended to serve as a comprehensive monograph on TRMS. Since the definition of TRMS (cf. Chapter 1) is broad, the scope of this book is extensive. In the first three chapters, we outline the main principles of mass spectrometry. We discuss common ion sources and mass analyzers emphasizing the features of these devices that make them useful in time-resolved measurements (Chapters 2 and 3). Subsequently, we introduce and discuss the design of instrumentation for such measurements considering sample delivery and treatment stages (Chapters 4–9). In Chapter 5, we highlight the trade-off between acquisition speed (temporal resolution), mass resolution, and sensitivity. Eventually, we enumerate examples of different detection/analysis schemes in TRMS that can be implemented in real-world applications (Chapters 10–13). Finally, we present the prospects for future developments and applications of TRMS (Chapter 14). The contents of this book are arranged in a logical sequence but individual chapters can also be read separately – not following the order of the table of contents. Some of the chapters provide a general background on mass spectrometry related technology (Chapters 2 and 3) while the others contain reviews of the previous achievements related to TRMS along with numerous references to original papers and specialized reviews (Chapters 4–13).

We hope this monograph can help science and engineering students better understand the concept of TRMS, and recognize the usefulness of dynamic monitoring of chemical and biochemical processes. We believe it will benefit researchers in academia (including research students and assistants) in the fields of chemistry, physics, and biology; students attending courses in analytical chemistry, organic chemistry, biochemistry, biophysics; as well as industrial scientists.

The field of TRMS is growing rapidly, and almost every month one can read about new exciting developments in the chemistry literature which take advantage of mass spectrometry as a tool to follow processes in time or to detect short-living species. Therefore, we suggest readers consider this book as a primer to TRMS but also recommend following the current progress in TRMS by reading recent articles published in peer-reviewed journals related to mass spectrometry and analytical chemistry. The authors welcome feedback from readers. We are keen to read comments and listen to criticisms. We also apologize to those authors of original papers, whose excellent mass spectrometry work has not been cited in this short monograph, or discussed to any great extent, due to space restrictions.

Pawel Lukasz Urban

Yu-Chie Chen

Yi-Sheng Wang

Acknowledgments

We wish to thank our talented co-workers with whom we interact on a daily basis. These interactions have certainly been beneficial to the writing of this book. Special thanks are due to Professor Yen-Peng Ho (National Dong Hwa University) and Dr Kent Gillig (Academia Sinica) for their comments on the book's manuscript. Yi-Sheng Wang would like to thank Professor Sheng Hsien Lin (National Chiao Tung Univeristy) and Dr Sabu Sahadevan (Bruker Taiwan) for useful discussions and comments. Any outstanding errors are the authors' responsibility.

List of Acronyms

Chapter 1

Introduction

Time flies over us, but leaves its shadow behind.

Nathaniel Hawthorne (1804–1864)

1.1 Time in Chemistry

According to one definition, time is the indefinite continued progress of existence and events in the past, present, and future regarded as a whole [1]. For millennia, time has intrigued philosophers and artists. The inevitability of time flow has triggered frustration and hope. While basic chemical knowledge was gathered in antiquity, modern chemistry has been developed since the 17th century. During the first c01-math-0001 years, the notion of time in chemistry was obscure and elusive. It appeared in the spotlight when Peter Waage and Cato Guldberg began to develop the concept of chemical kinetics. Since then, the time dimension was instantly promoted to become an important factor in chemical reactions. In modern chemistry textbooks, potential energy diagrams often represent changes in the energy of reactants along an axis labeled as the reaction path. However, time is the variable that describes the progress of every chemical transition and physical process. Thus, time has always been among the key factors studied in chemical science [2]. Investigating chemical phenomena in relation to time has turned out to be vital for the understanding of fundamental concepts – in particular, reaction kinetics.

Temporal resolution is the ability of a method to discern consecutive transitions in the studied dynamic systems. In the field of analytical chemistry, there exist numerous methods that allow one to measure concentrations of substances in solutions or gaseous mixtures at given time points. However, many conventional methods possess limited temporal resolution. In some cases, samples are obtained from reaction mixtures at specific time points. As a result the temporal characteristics of the studied process can only be described considering the limited frequency of sampling points. The obtained samples can be regarded as zero-dimensional. We live in a four-dimensional world that is described by three spatial dimensions. Time is the fourth elusive dimension that describes happenings in the other three dimensions (change of position). Various novel analytical methods have been developed to grasp the 3D nature of chemistry. For example, optical methods are irreplaceable when it comes to 2D and 3D spatial analysis (imaging) of chemical processes [3, 4]. Frequently these methods also provide superior temporal resolutions. Due to numerous technical obstacles, advancement of these multidimensional analysis tools could only happen because of the developments in physics, optics, photonics, and physical chemistry.

Some physical or chemical processes are so fast that their existence can only be verified using highly refined analytical approaches. For example, using infrared (IR) spectroscopy and computational methods, it was possible to confirm the existence of the simplest Criegee intermediate c01-math-0002 which has a lifetime counted in microseconds [5]. Other processes (e.g. radioactive decay of uranium-238 with a half-life of c01-math-0003 ) are so slow that their progress cannot easily be observed during a human lifetime. However, most reactions occurring in the biological world are accelerated by biocatalysts (enzymes). Such catalytic processes can be observed on timescales of seconds and minutes. Reaction kinetics encompasses experimental methodology and the associated mathematical treatment aiming to describe the progress of chemical reactions in time. Understanding chemical kinetics can help us to optimize important reactions, so that they can be applied in large-scale synthesis, and used by industry. The kinetic profiles of reactions let us gain fundamental insights on the reaction mechanisms. Similarly, to chemical reactions, there exist other processes which serve chemists every day – distillation and extraction are just two examples. Studying kinetic properties of dynamic processes involving molecules requires the use of appropriate analytical methodology – capable of recording molecular events in the time domain.

Several physical techniques were introduced to chemistry laboratories in order to enable monitoring of chemical and physical processes in time. They include such dissimilar platforms as: fluorescence detection [6], IR spectroscopy [7], diffraction [8, 9], nuclear magnetic resonance (NMR) [10–12] as well as crystallography [13]. Since ultrafast phenomena are relevant to many fundamental studies in physics and chemistry [14], various spectroscopic techniques have been developed which enable investigation of molecular events in the time range from 10-9 to 10-18 s [15, 16]. Ultrafast IR and Raman spectroscopies enable measurements of phenomena which occur on the pico- and femtosecond timescales c01-math-0004 ; corresponding to the elementary steps that affect chemical reactivity, including changes in the electron distribution, molecular structure and translocation of chemical moieties [17]. Pulse fluorometry and phase-modulation fluorometry enable the measurement of fluorescence lifetimes, which typically last over c01-math-0005 [18, 19]. Time-resolved luminescence methods are routinely used in fundamental and applied sciences. For instance, by monitoring the intensity of light emitted following an excitation pulse in the nano- to millisecond range, one can distinguish contributions of de-excitation of individual fluorophores and/or phosphors. This approach enables sensitive detection of labeled molecules (or supramolecular probes) in complex biological samples which possess intrinsic luminescence (e.g., autofluorescence). Lifetime spectroscopy is nowadays almost routinely used in the analysis and imaging of biological specimens [20].

Optical methods have grounded their place in chemistry. They also have intrinsic limitations: the most prominent one is low molecular selectivity. Monitoring unknown substances and identification of unknown analytes, which are frequently present in complex mixtures, often requires powerful analytical strategies. Two particularly significant ones are NMR and mass spectrometry (MS). These two techniques have dissimilar principles: while MS separates and detects ions in the gas phase, NMR recognizes nuclei based on the characteristic electromagnetic radiation emitted by them at specific conditions in a magnetic field. Due to its versatility in structure elucidation, it is certainly worthwhile studying the principles and applications of NMR in chemistry with the aid of recent monographs (e.g., [21, 22]).

1.2 Mass Spectrometry

MS is one of the main techniques used in chemical analysis nowadays [23–28]. Due to its capabilities in molecular identification and structure elucidation, as well as high sensitivity [29], it has already enabled important discoveries in chemistry and the biosciences. A mass spectrometer consists of three main parts: an ion source, a mass analyzer, and a detector (Figure 1.1). They are supplemented with a number of auxiliary components, including interfaces for sample pre-processing and introduction, ion optic elements for manipulating ion beams, and electronic data acquisition systems.

Figure 1.1 Main components of a mass spectrometer. Reproduced from Kandiah and Urban [29] with permission of The Royal Society of Chemistry

In the gas phase, it is generally easier to handle and separate ions than neutral molecules. Gas-phase ions are produced in the ion source (see Chapter 2), while the mass analyzer subsequently separates them according to their characteristic mass-to-charge ratios c01-math-0006 . There are various types of mass analyzers (see Chapter 3). For example, in a quadrupole mass analyzer, ions pass through a zone between two pairs of metal rods spaced radially along the ion propagation axis. The ratio of alternating and direct current electric fields produced in this section enables selection of ions with a specific c01-math-0007 value, and discrimination of other ions. A detector – positioned at the end of the quadrupole (on the side opposite to the ion source) – counts the ions which have passed through the quadrupole zone. In order to prevent collisions of gas-phase ions with gas molecules, analyzers and detectors are held under high vacuum. Some ion sources also operate under vacuum while in others the process of ion generation occurs at a higher pressure up to atmospheric pressure.

MS has evolved in the past few decades. There have been several milestone discoveries and inventions (Table 1.1). They encompass fundamental physical phenomena related to ion formation, as well as technical aspects – including the construction of ion sources and mass analyzers. Since the operation of these two components is crucial for the measurements conducted in the time domain, they will be discussed extensively in the following chapters.

Table 1.1 Selected milestones in MS

A mass spectrum is the primary result of mass spectrometric analysis. The pioneer of MS – Francis Aston – recorded mass spectra of separated ions by exposing a photographic emulsion to the ion stream: the resulting dark bands marked the regions corresponding to the beams with high ion intensities (Figure 1.2a). The positions of these bands along the horizontal axis of such a radiogram are related by the c01-math-0008 of the impinging ions: the darker the band the higher the intensity of the ion beam. Modern mass spectrometers use electronic sensors to measure the intensities of ion fluxes – in space, in time, or in both dimensions (depending on the design of mass analyzer). These intensities are classified into discrete channels. In the course of mass calibration, these channels are related by the m/z. As a result of this operation, a mass spectrum is produced (Figure 1.2b). It can be viewed as a histogram representing ion counts (intensities) in very narrow intervals of the m/z scale.

Figure 1.2 (a) Mass spectra obtained by Aston using the early mass spectrometer. Reproduced from Squires [50] with permission of The Royal Society of Chemistry. (b) Mass spectrum of caffeine obtained using a modern ESI-IT mass spectrometer. Courtesy of E.P. Dutkiewicz

1.3 Time-resolved Mass Spectrometry

In biochemistry it is often necessary to capture changes in the metabolic composition of samples (e.g., [30, 31]). For instance, animals communicate with each other chemically by releasing signaling molecules called pheromones, which persist in the proximity of an individual for a short period of time [32]. In the domain of exact sciences, it is also important to study temporal changes of chemical systems (reactions) and physical phenomena (e.g., molecular transport). Although many mass spectrometric analyses are often conducted as single-time-point measurements, it is evident from numerous reports published over the past few decades that MS can facilitate the studies of dynamic processes in which concentrations or structures of analyte molecules change over time. In particular, the technique enables structural determination of reactants while preserving temporal resolution [33]. While other techniques – notably, electronic and vibrational spectroscopy – can record time-dependent data, time-resolved mass spectrometry (TRMS) provides orthogonal information (m/z vs. abundance vs. time) to those of other established techniques. Since short-living reaction intermediates can be detected by various MS methods, this technique is ideal for studies on mechanisms of chemical reactions, chemical kinetics, and biochemical dynamics.

Dynamic phenomena have been investigated with MS for half a century now (e.g., [34–37]). Ions cannot move as fast as photons while mass spectrometers typically operate in the microsecond regime. Therefore, MS cannot equalize temporal resolutions achieved with some optical techniques (which are currently limited by the speed of detection systems rather than the speed of photons). Temporal resolution is a critical limitation when it comes to identifying reaction intermediates and measuring reaction kinetics by MS [38]. As it will be evident later on, the speed of MS is currently limited by the speed of sample processing rather than the speed of ions.

Here we define TRMS as a mass spectrometric approach that allows one to differentiate between two chemical states that can be observed sequentially at two points on the timescale. Such a broad definition encompasses various methods with quite dissimilar temporal resolutions and diverse areas of applications. A large variety of physical and chemical processes can be studied by TRMS: from protein folding, to enzyme kinetics, to mechanisms of organic reaction, extraction, and combustion [33]. Since short-lived reaction intermediates can be detected using several TRMS methods, this platform is ideal for the studies on mechanisms of chemical reactions, chemical kinetics, and biochemical dynamics. Depending on the focus area, temporal resolutions typically range from microseconds to minutes. While many organic reactions occur in hours, they do not require sophisticated setups to follow their kinetics or characterize intermediates (see Chapter 11). On the other hand, some of the studies on protein folding may require temporal resolutions below 1 ms (see Chapter 12).

The need to achieve satisfactory speed of MS analysis encouraged the development of delay-free sampling strategies. Early attempts to demonstrate TRMS date back to the 1950s, when radicals were recorded for thermally decomposing gases [34, 35], and flash photolysis reactions were captured using customized time-of-flight (TOF) instruments [36] (see also Chapter 4). In some cases, obtaining time-resolved data required construction of complicated experimental systems and solving cumbersome technical problems. Importantly, it is not always necessary to take a lot of effort to assemble complex apparatus since many pieces of hardware are already available commercially, and often minor modifications of these standard instruments are sufficient to carry out TRMS measurements. TRMS encompasses an assortment of methods based on different principles. Some TRMS systems are so dissimilar that the only common feature is the use of a mass spectrometer as the detector. Using customized apparatus, a multitude of physical, chemical and biochemical phenomena can be characterized in the time domain.

1.4 Dynamic Matrices

The concept of sample is one of the most widely used in analytical chemistry. According to the International Union of Pure and Applied Chemistry, sample is defined as a portion of material selected from a larger quantity of material [39]. Often we talk about representative samples which are supposed to reflect the chemical composition of the sampled medium at the time of sampling. However, many chemical media do not represent a constant chemical composition. Therefore, samples collected at different time points may have different components – qualitatively and quantitatively. For example, the chemical composition of river water may change in the course of rainfall or discharge of effluent from a wastewater treatment plant. Contents of a chemical reactor (reaction vessel) change as the reaction moves on, that is the concentrations of reactants decrease while the concentrations of products increase. If we continuously collect aliquots of media from such systems (river, reactor), and perform chemical analysis, we will find that the composition of the obtained fluid varies qualitatively and quantitatively. Therefore, in some cases it would be helpful to use the term dynamic sample (or dynamic matrix) to describe chemically unstable media that change with time. The concept of dynamic sample is in opposition to static sample (or steady sample) – which does not change its composition over time. However, in the following discussion we will also deal with cases in which spatial gradients of reaction products are formed downstream from micromixing devices (see Chapter 4). Generation of such gradients enables studies of some fast phenomena (e.g., protein folding measurements, see Chapter 12). Nonetheless, probing analytes from spatial concentration gradients does not rely on time-resolving capabilities of MS. Thus, the composition of an aliquot obtained from a fixed position within a spatial gradient can be referred to as quasi-static (or quasi-steady).

1.5 Real-time vs. Single-point Measurements

It is necessary to distinguish between two modes of recording temporal information in TRMS:

Real-time monitoring provides the means to record data continuously with a specified temporal resolution. Multiple spectra are recorded at a high frequency (typically, several spectra per second). Here, the spectral patterns are assumed to reflect the chemical compositions of the investigated systems at given time points. The consecutive spectra are expected to represent different patterns reflecting qualitative and/or quantitative changes in the investigated systems. The time interval between two consecutive analyses (data points) should be as short as possible in order to achieve high temporal resolution of real-time monitoring.

Single-point measurements provide spectral snapshots of the investigated systems at selected time points. If the system is dynamic or unstable, it may require quenching before introduction to the ion source. In this case, the time of quenching rather than measurement itself puts the time tag on the molecular composition of the dynamic sample. In some cases, one of the ionization steps (e.g., removal of solvent) may be regarded as quenching.

When investigating highly dynamic systems, the sampling time or the incubation time is an important attribute of MS measurements. Other chemical systems exist in equilibrium, in which case steady samples are obtained, and the sampling time becomes a less important descriptor. As mentioned above, in some designs of TRMS, reaction mixtures are generated continuously in the flow of the reactant stream or droplet plume. In those cases, quasi-steady samples are produced. Thus, single-point measurements of reaction products or intermediates can be conducted following a very short incubation period.

1.6 Further Reading

The reference lists included in every chapter contain numerous examples of representative reports on TRMS. For more succinct summaries of TRMS, as well as the coverage of specific topics, readers are also encouraged to consult recent review articles and chapters [33, 40–46]. While some of the following chapters cover important parts of MS workflow, readers interested in more general aspects of the technique are encouraged to consult MS textbooks [23–28]. Those who are specifically interested in the application of MS in the studies of reaction intermediates of chemical reactions, and would like to gain a more extensive overview of that subject, are encouraged to read the book edited by Santos [47].

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