Applications of Modern Mass Spectrometry: Volume 2 E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Serie: Applications of Modern Mass Spectrometry
- Sprache: Englisch
Applications of Modern Mass Spectrometry, Volume 2, covers the latest advances in mass spectrometry in scientific research. The series presents readers with information on the broad range of mass spectrometry techniques and configurations, data analysis, and practical applications. Each volume contains contributions from eminent researchers who present their findings in an easy-to-read format. The multidisciplinary nature of the works presented in each volume of this book series makes it a valuable reference on mass spectrometry to academic researchers and industrial R&D specialists in applied sciences, biochemistry, life sciences, and allied fields.
The second volume of the series presents 6 reviews: Ion Mobility-Mass Spectrometry for Macromolecule Analysis - Recent Advancements in Detection of Organic Contaminants in Wastewater Using Advanced Mass Spectrometry - Poisonous Substances in Tropical Medicinal and Edible Plants: Traditional Uses, Toxicology, and Characterization by Hyphenated Mass Spectrometry Techniques - LC-MS Analysis of Endogenous Neuropeptides from Tissues of Central Nervous System: An Overview - Advances in Structural Proteomics Using Mass Spectrometry and Recent Trends of Modern Mass Spectrometry: Application towards Drug Discovery and Development Process.
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Veröffentlichungsjahr: 2024
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PREFACE
Mass spectrometry is a unique analytical tool that offers unmatched sensitivity and selectivity levels for a wide range of analyses. The most recent applications of mass spectrometry are mostly oriented toward biochemical problems, such as proteomes, metabolomes, high-throughput drug discovery, and metabolism. Other analytical applications are routinely applied in pollution control, food control, forensic science, natural products or process monitoring, and many others.
The present volume of “Application of Mass Spectrometry” provides a useful insight into some of these developments. The present 2nd volume of this book series comprises 6 comprehensive reviews written by the leading practitioners of mass spectrometry. These articles present diverse applications of mass spectrometry in fields such as proteomics, peptidomics, drug development and discovery, toxicology, and environmental analysis. Moreover, the use of advanced ionization techniques, i.e., ion mobility, particularly in the analyses of macromolecules, is also discussed in this volume.
Mehmet Atakay et al. have discussed the use of an advanced mass spectrometry approach, ion mobility spectrometry (IM-MS), in the field of macromolecule analysis, such as proteomics, glycoproteomics, and polymer characterization. Sarah Otun et al. have described the use of different mass spectrometric approaches and tools in the understanding of structural proteomics. Neva Alasağ et al. have elaborated on the use of liquid chromatography-mass spectrometry (LC-MS) as a powerful analytical technique for separating and quantifying endogenous neuropeptides in the central nervous system (CNS) and organisms. Louis P. Sandjo et al. have focused on the separation and detection of toxic plant-based metabolites in tropical medicinal and edible plants. Shweta Sharma has reviewed the use of mass spectrometry in various stages of the drug discovery and development process, including target identification, hit identification, lead optimization, and drug metabolism and pharmacokinetic studies. Imalka Munaweera et al. highlighted the qualitative and quantitative detection of a diverse range of organic contaminants in environmental samples utilizing advanced mass spectrometry.
We are grateful to all the authors for their excellent scholarly contributions and for the timely submissions of their review articles. We would also like to express our gratitude to Mrs. Fariya Zulfiqar (Manager Publications) and Mr. Mahmood Alam (Director Publications) of Bentham Science Publishers for the timely completion of the volume in hand. We sincerely hope that the efforts of the authors and the production team will help readers better understand and appreciate the versatility and robustness of mass spectrometry and motivate them to conduct good-quality research and development work in this exciting area.
List of Contributors
Ion Mobility-Mass Spectrometry for Macro- molecule Analysis
Mehmet Atakay1,Hacı Mehmet Kayılı2,Ülkü Güler1,Bekir Salih1,*Abstract
The need for conformational information is increasing by the time in studies on macromolecules. For example, proteins may have various functions and properties depending on their folding states that make their conformational analyses very important. Mass spectrometry is one of the most effective analytical techniques that separate ions in the gas phase by their mass-to-charge ratio. It provides useful data on molecular characterization in many areas of research with high precision, accuracy, and sensitivity. Although mass spectrometry is a very powerful analytical technique, it cannot distinguish different species having identical mass-to-charge ratio. The analytical technique combining mass spectrometry with ion mobility spectrometry (IM-MS), which provides information about the three-dimensional structure of an ion, solves this problem by separating them according to their collision cross sections (CCS) in the gas phase. This analytical method also provides the advantages of higher precision and better resolution in the rapid analysis of different types of complex samples. The separation of isomers with the same molecular weight, increasing the dynamic range and distinguishing ions from chemical noise are the most important features that this technique contributes to mass spectrometry. As improvements have been made in IM-MS technology, the number and quality of publications in the areas where this technique is used increases rapidly. In this chapter, the use of IM-MS techniques in the fields such as proteomics, glycoproteomics and polymer characterization are explained by presenting their various applications in the literature.
INTRODUCTION
The status and use of ion mobility-mass spectrometry (IM-MS) in various research areas have been increasing rapidly in recent years [1]. The interest in this analytical method is increasing in parallel with the improvements in the parameters such as high sensitivity, resolution power and low amount of sample
in the analysis with the developing technology. The basics of ion mobility spectrometry are similar to the principles of mass spectrometric techniques. Thus, these two techniques can be easily used together in a combined system. Modern IM-MS techniques having high ion mobility resolution, sensitivity and applicability for a wide variety of samples started to be developed in the 1990s. These developments coincide with the period when ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) were first used in mass spectrometric analysis [2-4]. In 2006, a company manufacturing mass spectrometers combined ion mobility and mass spectrometry techniques in an instrument and launched it on the market. Since then, the use of ion mobility technology has become increasingly common in various research areas for differentiation, identification, and structural analysis of species [5]. Today, commercially available IM-MS instruments having different ion mobility technology provided by different manufacturers are used in studies [1].
Ion mobility spectrometry is still widely used alone in defense, security and environmental analysis applications [6-8]. Over the past two decades, very rapid and significant advances have been made in the development of systems in which ion mobility and mass spectrometry are combined. These advances are particularly concerned with the ability to trap, transmit and focus ions between regions under different vacuum values using electrodynamic fields. Considering the very important analytical advantages provided by IM-MS instruments, ion mobility technique has become a preferable option especially in studies carried out in the “omics” fields [9]. IM-MS technique, which has an important place in various research fields, has become an analytical system that is also sought after and interested in several applications performed on the industrial scale.
Ion mobility spectrometers simply measure the time spent by ions as they travel through an electrical field with the help of a buffer gas. Ion mobility (K), collision cross section (Ω), the Boltzmann constant (kb), neutral number density (N), mass of ion (m1), mass of buffer gas (mN), charge of ion (z), and electronic charge (e, 1.602 x 10-19 C) is defined by the Mason-Champ equation [10, 11].
(1)The collision cross section (CCS) value of an ion can provide detailed information about its size and shape. When two different molecules having identical mass-to- charge ratios are analyzed using IM-MS, they can be separated from each other according to their mobility in the ion mobility cell depending on their shape and size characteristics. The arrival time data obtained from the acquisition with an IM-MS device should be converted to the CCS values of the analyzed species according to the performed calibration calculations using standard molecules with known CCS values. There are free software and databases that can be helpful in such CCS calibration and estimation processes. A list of common CCS databases and software used in CCS calibration, prediction, and estimation with their URLs are given in Table 1.
The collision cross section of an ion depending on its mobility and the type of buffer gas used can also be calculated by appropriate computational methods. Thus, the theoretical CCS values can be compared with the experimental values obtained from IM-MS analyses. The theoretical average CCS values are determined by taking into account the collisions of ions with the buffer gas in the ion mobility cell by the computational simulation of analysis conditions [12]. In structural biology studies, the conformations of biomolecules or their complexes are investigated by comparing the experimental CCS values with data obtained from theoretical calculation [13].
In this chapter, the structural analysis of proteins, peptides, glycoconjugates, and polymers, as well as the applications of analyzing the conformational features of these macromolecules using ion mobility-mass spectrometry is reviewed. The capability of the IM-MS technique is explained by what kind of structural data and information can be obtained about the conformational features of these groups of species.
IM-MS Analysis in Proteomics
The data obtained from researches involving proteomics approach in clinical applications such as diagnosis of diseases, treatment and enlightening of their molecular mechanisms are increasingly important [45, 46]. Proteomics plays a major role in monitoring many diseases in recent years. However, analyses performed in this field still lack of deficiencies in the detection and quantification of low abundance proteins such as transcription factors, signaling agents or emergent protein products of genomic problems [47].
Proteomics is divided into two groups, which are the “bottom-up” and “top-down” approaches. In the bottom-up approach, proteins can be identified by analyzing the resulting peptide mixture obtained after the enzymatic cleavage. In the bottom-up strategy, proteins are digested and then identified based on their specific peptides. In the top-down approach, intact proteins are identified directly with the help of high resolution and tandem mass spectrometry (MS/MS) capabilities of mass spectrometers. In the bottom-up approach, it is necessary to perform chromatographic separation combined with mass spectrometers that can provide higher sensitivity for complex samples. Thus, it is possible to differentiate the data originating from the multiply charged and isobaric species. In some instances, even in analysis with (liquid chromatography-tandem mass spectrometry) LC-MS/MS systems that allow high resolution and sensitivity, many peptides in very low amounts cannot be detected in a single run analysis. Herein, the hyphenation of the ion mobility spectrometry technique into LC-MS/MS analysis methods may provide much more detailed data for proteome samples by distinguishing the signals from such low amounts of peptides [48].
In the first applications of IM-MS technique in the field of proteomics, conformational characterizations of some specific peptides were made by calculating their CCS values [34]. In subsequent studies, research groups used the IM-MS technique in the proteome sample analysis to separate the peptide isomers based on their slight structural differences and calculate the CCS values of the peptides in the samples containing a large number of peptides [49-58]. The advantages obtained by IM-MS technique provides detailed information about the functional mechanisms of biological systems as a result of obtaining much more comprehensive data in the analysis of enzymatic digestion products of proteins in biological samples.
Fig. (1)) Bottom-up proteomics LC-IM-MS and LC-IM-MS/MS workflows.By incorporating the ion mobility technique into standard LC-MS analyzes, the peptides can be identified with much higher resolution, increasing the data capacity from MS/MS analysis results [59-63]. If such advantages obtained from IM-MS technique can be combined with a good separation method and chemical labeling process in proteomics studies, protein identification and conformational analysis of protein complexes can be improved by enabling the vast majority of species to be identified [61, 64-66]. Alternating LC-IM-MS and LC-IM-MS/MS workflow options in bottom-up proteomics approach are shown in Fig. (1).
In bottom-up proteome analysis, species are generally analyzed with targeted and shotgun proteomics based approaches. However, it can be seen that species can be identified more comprehensively by combining these approaches with discovery and targeted monitoring (DTM) method [47, 67]. Kristin E. Burnum-Johnson et al. stated a method that contains DTM in a single analysis by combining both LC-MS and IM-MS techniques (LC-IM-MS). The DTM method provides much more peptide coverage compared to LC-MS analyzes where long method times are applied by allowing identification of lower abundance species [47]. In the DTM method, heavy labeled target peptides are generally added to the peptide mixture obtained by tryptic digestion and both labeled and unlabeled peptides are analyzed simultaneously with the LC-IM-MS technique. In this method, heavy-labeled peptides are added to the enzymatic digest mixture for quantification of peptides while unknown peptides are also identified in multiple dimensions according to their retention times, CCS values and exact masses. With this technique, very low amount of species can be analyzed with high sensitivity and resolution, so that the higher peptide and protein scores can be reached and very detailed information about the sample can be obtained.
It seems that the use of IM-MS provides a great advantage for the analysis of isomeric peptides that are difficult to separate by conventional methods such as liquid chromatography. Isomeric peptides have different amino acid sequences while their amino acid compositions are identical. In addition, the peptides containing isomeric amino acids (i.e. leucine and isoleucine), presence of various post-translational modifications located on different amino acid sites of the peptide, and the conformational variations of the peptides such as D- and L- may cause the presence of isomeric peptides in the samples. Mass spectrometers including mass analyzers such as fourier-transform ion cyclotron resonance (FTICR) or Orbitrap which provide the highest resolution values are used in LC-MS /MS analyses to obtain detailed data in bottom-up targeted proteomics studies. In proteome analyzes with such instruments, which provide the highest mass resolution, longer chromatographic separation time should be performed to distinguish the peptides in complex biological samples. It is very difficult to separate isomeric peptides that have very similar chemical properties, even if very long chromatographic methods are performed in such analyzes. Although the instruments provide high mass resolution, isomeric species with the same mass-to-charge ratio cannot be distinguished by single-step mass spectrometric analysis. Even by using MS/MS techniques such as collision-induced dissociation (CID), comprehensive amino acid sequence analysis cannot be performed using the data obtained for isomeric parent ions. The unique fragmentation methods such as electron capture dissociation (ECD), electron transfer dissociation (ETD) and ultraviolet light photodissociation (UVPD) have been used in amino acid sequence analyses recently. However, these methods may also have various limitations such as low compatibility with the instruments and commercial accessibility. In such proteome analyses, it is possible to obtain more detailed data by using IM-MS technique compared to other analysis methods, by reducing the analysis time and increasing the probability of differentiation of species [68].
The use of IM-MS technique in the fields of structural biology and proteomics has become preferable, as a result of the necessity of revealing the important link between the conformations and functions of biomolecules. The IM-MS technique could be used successfully in structural biology and intact proteomics studies especially on neurodegenerative diseases such as Alzheimer and Parkinson in which some of the protein structures disordered or change [13, 69-81]. In such diseases where deterioration in the conformation of proteins causes accumulation in tissues, the progression mechanisms of the disease can also be monitored at the molecular level by using the IM-MS technique. Recent studies in the field of intact proteomics the structural stability and conformation of proteins or their complexes are investigated by applying variable collision voltage using collision-induced unfolding technique [82].
Differential ion mobility spectrometry (DMS) is also used for conformational analysis of peptides and proteins in proteome samples. The ion mobility separation mechanism of DMS technique has high compatibility with mass spectrometers. In the analysis made using DMS technique, the ions can be separated in the ion mobility unit of the instrument according to their shape and charge features prior to mass spectrometric analysis. By using the hyphenated DMS-MS technique, rapid and detailed analysis of complex protein and peptide samples that may have variable structure as a result of post-translational modifications is possible. In the vast majority of proteomics studies, post-translational modifications in protein structures and the relationship of these changes with protein functions are intensely studied. Using the DMS technique, isobaric peptides, which are quite difficult to separate from each other by other analytical methods, can be identified by distinguishing them according to the position of functional groups in their chemical structure. Highly complex proteome samples can be analyzed using the tandem ion mobility (IM/IM) technique including DMS technique, which provides conformational separation in multiple dimensions. Thus, very detailed information can be obtained among the analyses of proteome samples by acquiring the data from this analytical technique. It may be useful to create and use standard databases for data that can be obtained from IM/IM analysis, such as data and spectra in standard databases used in MS/MS analyses in the field of proteomics [83, 84].
In some studies, important information about proteome samples can be obtained by combining the data obtained from bottom-up proteomics approach and intact protein analysis. In order to structurally analyze proteins and their complexes by IM-MS, it can be very useful to have protein identification data as supporting. The conformation information obtained from the analysis of protein complexes performed with the IM-MS technique can be supported by the results obtained from the bottom-up analysis of the complex constituent and the top-down analysis of the denatured proteins. The data obtained from IM-MS analysis provides more detailed data not only in the clarification of protein conformations, but also in peptide mass determination, amino acid sequence analysis and analysis of the components of protein complexes [49, 85].
Peptide biomarkers in very low concentrations in the mixture containing untargeted peptides could be detected using trapped ion mobility spectrometry (TIMS) technique, which provides the highest resolution value in ion mobility-mass spectrometers used in the field [67].
In recent studies, it has been shown that complex proteome samples can be analyzed with high coverage with the “Parallel Accumulation - SErial Fragmentation” (PASEF) method, which synchronizes TIMS with the parent ion selection process in MS/MS technique [52, 86, 87]. In this method, it was determined that analysis speed and performance increased as a result of minimizing loss of sensitivity by allowing multiple parent ions to be fragmented in each TIMS scan. In PASEF technique, as the parent ions are accumulated, the disadvantages sourced from fast MS/MS analyses can be minimized, and the loss of ions can be prevented. The instrument including the PASEF concept is named Bruker timsTOF Pro and its performance is primarily evaluated in the field of proteomics [48]. Some research groups are working on the evaluation of the data obtained from timsTOF Pro analyses using the MaxQuant program.
In these studies, MaxQuant version with CCS values is tried to be developed into the version being used for LC-MS/MS data. It is stated that evaluating IM-MS data with a currently used program such as MaxQuant may increase the consistency and relevance of proteome analyzes. By integrating the MS/MS spectra, retention time, and mobility values of the species in the MaxQuant program, it is thought that very useful results can be obtained for the identification of proteins and label free quantification applications [88, 89].
IM-MS Analysis of Glycoconjugates
Glycans are crucial biomacromolecules consisting of different monosaccharides. They are conjugated to proteins or lipids and, these glycoconjugate structures can be located on the cell surfaces. They involve in many important cellular events such as cell-cell interactions, cell recognizing and immune response [90]. Therefore, it is important to characterize their structures with analytical methods for understanding the functions of them. However, the complexity of their structures makes the analysis very challenging and problematic.
It can be observed different linkages between two monosaccharides. This cause to monitor possible stereoisomers at the anomeric carbon of one monosaccharide [91, 92]. In addition, regioisomerism can be monitored on the sugars because of hydroxyl groups that are occupied different positions in a monosaccharide. These isomers are of course showed different three-dimensional conformations. On the other hand, a monosaccharide residue can attach to two other monosaccharides by doing two glycosidic bonds, which gives rise to increase structural complexity of the glycans. In addition, glycan isomerization can be observed on glycans based on binding region of a monosaccharide residue. For example, fucose can locate on glycans either by their core or branch regions [93]. Glycans can be found in different forms onto the proteins and classified based on their branching types and monosaccharide contents (Fig. 2).
Fig. (2)) Main N- and O-glycan types are illustrated.Main Strategies for the Analysis of Glycoconjugates by Mass Spectrometry
Mass spectrometry can be commonly used for the analysis of glycans. Glycans can be analyzed by mass spectrometry at two level. In the first level, glycans can be released from their glycoconjugate domains such as proteins and lipids either by an enzymatical or a chemical process. This strategy is called as released glycan level. In the second strategy, glycan analysis is performed using glycopeptides derived from glycoproteins via proteolytic digestion, which is called as glycopeptide level. Both strategies are complementary and provided structural information about the glycans.
In the released glycan analysis, several chemical derivatization methods applied onto glycans can be performed to characterize N-glycan isomers prior to their MS analysis. For instance, ethyl-esterification approach allows to separate alpha-2,3 and alpha-2,6 sialic acid linkage positions for MALDI-MS [94-96]. However, the number of the high-throughput methods are limited for this purpose and do not cover all glycan isomers. Moreover, this type chemical procedures for identifying isomeric glycan structures contain additional sample preparation steps. On the other hand, a variety of chromatographic techniques such as hydrophilic interaction liquid chromatography (HILIC) and porous graphitic carbon chromatography (PGC) can be equipped with mass spectrometers, which enable to separate isomeric mixtures of glycans based on their retention times [97]. The main problem of these methods is that they cannot separate isomeric ions having the same mass and retention times.
Ion mobility-mass spectrometry (IM-MS) is provided to additional dimension for the characterization of glycans by performing a gas-phase separation before MS analysis [98]. Unlike the conventional mass spectrometers, IM-MS enables the separate glycans based on their size, shape and charge. This feature can also be utilized to decrease the mass spectra complexity of the obtained data [99, 100]. In addition, collision cross section (CCS) values of intact glycans can be obtained by the measurement of drift time. This property may use to elucidate glycan structures [101].
IM-MS Analysis of Intact Glycopeptides
Linkage-specific separation of sialic acid isomers has been widely analyzed by IM-MS. This strategy allows to discriminate alpha-2,3 and alpha-2,6 linked sialic acid containing N-glycan structures without a chemical derivatization technique. Recently, a methodology was described to characterize these sialic acid containing isomers using differential mobility spectrometry [102]. Similarly, site-specific mapping of sialic acid linkage isomers by IM-MS was demonstrated using proteolytic digests of standard glycoproteins [103]. However, this isomer separation was not achieved by IM-MS at the intact glycoprotein level and the glycopeptide level [104]. IM analysis of the acquired MS/MS fragments of the sialylated intact glycopeptides was allowed to identify sialic-acid isomers [104, 105]. The fragment of the oxonium ion including galactose (Gal), N-Acetylglucosamine (GlcNAc) and N-Acetylneuraminic Acid (NeuAc) that was formed by CID fragmentation showed different drift times based on linkage positions of NeuAc on the glycan fragment [103, 105]. Fig. (3) showed the workflow followed by these studies. It was determined that alpha-2,6 containing fragment displayed shorter drift times when compared with alpha-2,3 equivalent. In addition, the calculated CCS values for these fragments was found to be different, indicated that these values were diagnostic for the linkage-specific determination of NeuAc containing glycans [101].
Fig. (3)) A workflow scheme for linkage-specific analysis of sialic acid containing glycopeptides by IM-MS is illustrated. Scheme was modified from reference [105].One of the other applications of IM-MS for the isomeric characterization of glycopeptides was that it could be utilized to detect glycan attached site on the peptide part of a glycopeptide. By this approach, it was differed the location of N-Acetylgalactosamine (GalNAc) residues located on the same peptide sequence containing glycopeptides [106, 107]. These IM-MS approaches including CID and ETD can aid to detect glycosylation sites for O-glycosylation regions.
IM-MS Analysis of Intact Glycans
Glycans are usually analyzed using HILIC and PGC techniques equipped with MS or fluorescence detector (FLD). In this approach, released glycans can be tagged with different fluorophores and analyzed by MS and FLD containing instruments. IM-MS can be equipped with this approach for the analysis of glycan structures. Because ion mobility provides additional dimension, intact glycan analysis can be performed at four dimensions including retention time, intensity, mass-to-charge ratio and ion mobility. A recent study was described a workflow using HILIC-UPLC-FLD-IM-MS for the analysis of fluorescently labelled glycans. It was indicated that the assignment accuracy of the glycans increased thanks to glycan CCS values compared to conventional HILIC-FLD-MS analysis [108]. On the other hand, it was stated that fluorescent labelling influenced the separation of the glycan isomers [109]. In addition to HPLC-HILIC platform, capillary zone electrophoresis was used and evaluated for the analysis of isomeric glycans. This strategy was combined the separation based on electrophoretic mobility and ion mobility in a single platform [110].
Comparison of the methods for the separation of isomeric O-glycans by IM-MS and PGC techniques was demonstrated in a study [111]. It was stated that combination of these methods in a single platform would be robust for the recognition of full glycan profiles of complex samples. Indeed, important problems encountered for the interpretation of glycan structures could be eliminated. For instance; exact fucosyl positions in glycans could be found by IM-MS and thus, misinterpretation of MS/MS spectra belonging to fucosylated glycans was prevented [112]. Furthermore, information about the location of fucose residues was determined from specific ions related to each antenna [113].
Evaluation of IM-MS Platforms in Isomeric Glycan Analysis
Travelling-wave and drift tube ion mobility techniques are the most used platforms for isomeric glycan analysis. Most of the glycan types including high mannose, hybrid and complex type N-glycans has been worked with both platforms [114-116]. In addition, collision cross sections of the detected glycans were calculated using these platforms [115, 117, 118]. Moreover, a CCS database including estimated CCS data obtained from glycans and oligosaccharide standards by travelling-wave and drift tube ion mobility platforms was presented [16]. Recently, a serpentine ultralong path with extended routing (SLIM SUPER) IM-MS platform was employed for the characterization of isomeric glycans [119]. This technique provided better resolution and improved sensitivity when compared with conventional IM systems [120]. Similarly, selected accumulation or gated-trapped-ion mobility (SE/Gated-TIMS) equipped with fourier transform ion cyclotron resonance mass spectrometry platforms (FTICR-MS) was recently applied to analyze the isomeric glycans and sulfated glycosaminoglycans [121, 122]. In a study, a recently developed cyclic ion mobility (cIM) separator embedded in a quadrupole/time-of-flight mass spectrometer (QTOF) were assessed for the separation of isomeric pentasaccharides [123]. Besides these ion mobility platforms, differential ion mobility spectrometry (DMS) were employed for the isomeric characterization of sialic acid containing glycans [102]. A brief list of the studies including the glycan source, method, used platforms, and CCS estimation information were given in Table 2.
Isomeric separation of glycans derived from different glycoconjugate sources with different ion mobility platforms have been utilized both at the released glycan level and the glycopeptide level. By the combination chromatographic and electrophoretic techniques with IM-MS platforms, the glycan analysis can be achieved at fourth dimensions including retention time, intensity, mass-to-charge ratio and ion mobility. The calculated CCS values for each isomeric glycans can enhance the analysis efficiency. Therefore, these platforms would be valuable tools for in-depth characterization of isomeric glycans.
Polymer Characterization using IM-MS
Researches are being carried out intensively in the field of polymer science and add so many innovations to the literature at the present time. Although there are very important and rapid developments in the analysis of complex samples in this field, it is necessary to perform very challenging and costly studies in order to be able to characterize large synthetic polymer structures quickly, in detail with high accuracy. Polymers can have a wide variety of structural features, such as linear chain, ring or branched structure, homopolymer or copolymer, dendrimer and star polymers, each of which may have different length of arms.
Characterization of synthetic polymers should be carried out as extensively as possible to accurately determine their physical and chemical characteristics, which directly affect the applications of these polymers. In such studies, it is very important to conduct detailed analyzes on the important parameters that determine the physical and chemical properties of the materials in order to designate the functional properties of the polymers. The functionalities of many polymeric materials in biological and medical applications as well as general material science fields can be determined by monitoring the structural features of polymers using versatile analytical techniques [124-126]. The fact that the synthesis processes are difficult and the structures are required to be characterized by fast and reliable analytical methods that provide data with high precision and accuracy. As the molecular complexity in polymer samples increases, the need for an analytical technique such as mass spectrometry, which enables identification of species with high precision and accuracy, has emerged.
Materials consisting of linear or branched copolymers or cross-linked polymer structures in different molecular mass ranges and compositions are used in many applications. The determination of the repeating unit sequences of copolymers and conformational features of the polymer chains with high accuracy is of great importance in terms of confirming the usability of the materials produced in the field. Polymers can have different conformational properties such as cyclic, star-shaped, brush-type or dendritic. Complexed macromolecules, ion clusters, amphiphilic block copolymers and polyion complexes with such different conformations are used as encapsulation units in advanced tumor-targeted applications [127]. As the conformational complexity of the synthesized polymer increases, the synthesis process becomes more difficult and increases the probability that the intended material is faulty or incomplete.
Studies have been carried out since the early 2000s where polymer samples are structurally characterized by IM-MS techniques [128-130]. A list of some studies on polymer characterization using IM-MS techniques is given in Table 3 that shows the type of analyzed polymer, performed IM-MS technique and the interested molecular weight range in the referred studies.
In the characterization of the polymers, analytical studies are generally carried out on the calculation of Mn, Mw, and polydispersity index values, the identification of the chemical composition of the repeating units, side chains, end groups, the sequence of the copolymer chains, and the determination of the presence of impurities and additives [172]. With these factors, the structural stability of polymeric molecules and their interactions with other species are generally affect the conformational features of polymer chains that can be monitored by performing IM-MS analyzes (Fig. 4). The dynamic conformations of polymer chains have crucial importance on adjusting of their physical and chemical properties in various application fields.
Fig. (4)) Determinant effects on the conformations of polymers, which can be monitored by IM-MS.It is aimed to develop unique and new ionization techniques especially for polymer analysis using IM-MS technique. The researchers conducted IM-MS analyzes using the matrix-assisted vacuum ionization (MAIV) method they developed. Thus, the additives in the polymer samples could be determined in low amounts with the reduction of the noise in the gas phase [173]. In another study, poly (ether ether ketone) samples with low molecular mass were analyzed with the IM-MS system using the ionization technique called atmospheric solid analysis probe (ASAP). The ions obtained from this technique were found to be different from the ions obtained from MALDI-TOF analysis for the same samples [152].
By using the IM-MS technique, the conformational diversity of the structures formed by the dendrimers and linear polymer chains is examined [174]. Changes in the structures according to the amount of metal ions and charges added to the molecules could be monitored. The interpretation and evaluation studies were carried out by determining that the species had different drift times in the IM-MS analysis of the polymer sample containing isobaric linear and cyclic poly (caprolactone) chains [139]. In another study, polybutylene adipate oligomers ions carrying alkali metal cations (Li, Na, K, Rb, and Cs) as cationizing agent were characterized in detail using IM-MS, IM-MS/MS, and statistical analysis. It was concluded that the conformational features of polymer were not specific to either the cationizing agents or the instrumentation used in the study [167]. The structural trends of polymers could be compared according to the type of functional groups in their repeating units and end groups using data obtained from IM-MS analyses in several studies [166, 175]. The conformations of peptide-polyethylene glycol conjugates were analyzed in a study using IM-MS. The IM-MS analyses revealed the presence of random coil and helical conformers in both the peptides and conjugates. The IM-MS data also provided information that the helical structures of peptides are stabilized by polyethylene glycol (PEG) attached on their C-terminus [163].
