Ion Mobility Spectrometry Technique Research Articles

MobCal-MPI 2.0: an accurate and parallelized package for calculating field-dependent collision cross sections and ion mobilities.

Ion mobility spectrometry (IMS), which can be employed as either a stand-alone instrument or coupled to mass spectrometry, has become an important tool for analytical chemistry. Because of the direct relation between an ion's mobility and its structure, which is intrinsically related to its collision cross section (CCS), IMS techniques can be used in tandem with computational tools to elucidate ion geometric structure. Here, we present MobCal-MPI 2.0, a software package that demonstrates excellent accuracy (RMSE 2.16%) and efficiency in calculating low-field CCSs via the trajectory method (≤30 minutes on 8 cores for ions with ≤70 atoms). MobCal-MPI 2.0 expands on its predecessor by enabling the calculation of high-field mobilities through the implementation of the 2nd order approximation to two-temperature theory (2TT). By further introducing an empirical correction to account for deviations between 2TT and experiment, MobCal-MPI 2.0 can compute accurate high-field mobilities that exhibit a mean deviation of <4% from experimentally measured values. Moreover, the velocities used to sample ion-neutral collisions were updated from a weighted to a linear grid, enabling the near-instantaneous evaluation of mobility/CCS at any effective temperature from a single set of N2 scattering trajectories. Several enhancements made to the code are also discussed, including updates to the statistical analysis of collision event sampling and benchmarking of overall performance.

High-resolution ion mobility spectrometry-mass spectrometry for isomeric separation of prostanoids after Girard's reagent T derivatization.

Isomeric separation of prostanoids is often a challenge and requires chromatography and time-consuming sample preparation. Multiple prostanoid isomers have distinct in vivo functions crucial for understanding the inflammation process, including prostaglandins E2 (PGE2 ) and D2 (PGD2 ). High-resolution ion mobility spectrometry (IMS) based on linear ion transport in low-to-moderate electric fields and nonlinear ion transport in strong electric fields emerges as a broad approach for rapid separations prior to mass spectrometry. Derivatization with Girard's reagent T (GT) was used to overcome inefficient ionization of prostanoids in negative ionization mode due to poor deprotonation of the carboxylic acid group. Three high-resolution IMS techniques, namely linear cyclic IMS, linear trapped IMS, and nonlinear high-field asymmetric waveform IMS, were compared for the isomeric separation and endogenous detection of prostanoids present in intestinal tissue. Direct infusion of GT-derivatized prostanoids proved to increase the ionization efficiency in positive ionization mode by a factor of >10, which enabled detection of these molecules in endogenous concentration levels. The high-resolution IMS comparison revealed its potential for rapid isomeric analysis of biologically relevant prostanoids. Strengths and weaknesses of both linear and nonlinear IMS are discussed. Endogenous prostanoid detection in intestinal tissue extracts demonstrated the applicability of our approach in biomedical research. The applied derivatization strategy offers high sensitivity and improved stereoisomeric separation for screening of complex biological systems. The high-resolution IMS comparison indicated that the best sensitivity and resolution are achieved by linear and nonlinear IMS, respectively.

Study of atmospheric pressure chemical ionization of phthalates in air by ion mobility spectrometry/mass spectrometry.

Phthalates are widely used in consumer products in the chemical industries. Due to their abundance in the milieu, their potentially harmful effect on the environment, human and animal health there is a need for sensitive and fast methods for their detection. Positive polarity Corona Discharge Atmospheric Pressure Chemical Ionization (CD-APCI) in the air was applied for ionization of phthalates. The ionization method for the phthalates was studied by atmospheric pressure Ion Mobility Spectrometry (IMS) and hybrid IMS/orthogonal acceleration Time-of-Flight Mass spectrometry (IMS-oaTOF-MS). CD-APCI IMS and MS spectra of selected phthalates (dimethyl phthalate, diethyl phthalate, diethyl isophthalate, diethyl terephthalate, dipropyl phthalate, diisopropyl phthalate, dibutyl phthalate, diisobutyl phthalate, and dibutyl terephthalate) were recorded. In the case of the ortho- and "iso"-isomers exclusively the protonated molecular ions [M+H]+ were detected. In the case of the para- and meta-isomers and regioisomers, APCI resulted in the appearance of hydrated protonated molecular ions [M + H]+ ·(H2 O)0,1,2 . The ion mobilities, collision cross-sections of these ions in air, as well as the limits of detection (LODs) for the phthalate vapors, were determined. In the case of isomeric phthalates, we have demonstrated the potential of the IMS technique for their separation. The results show that CD-APCI in combination with IMS and IMS-oaTOF-MS is a suitable method for the fast and sensitive detection of phthalates with the potential to separate some isomers.

Determining Collision Cross Sections from Differential Ion Mobility Spectrometry.

The experimental determination of ion-neutral collision cross sections (CCSs) is generally confined to ion mobility spectrometry (IMS) technologies that operate under the so-called low-field limit or those that enable empirical calibration strategies (e.g., traveling wave IMS; TWIMS). Correlation of ion trajectories to CCS in other non-linear IMS techniques that employ dynamic electric fields, such as differential mobility spectrometry (DMS), has remained a challenge since its inception. Here, we describe how an ion's CCS can be measured from DMS experiments using a machine learning (ML)-based calibration. The differential mobility of 409 molecular cations (m/z: 86-683 Da and CCS 110-236 Å2) was measured in a N2 environment to train the ML framework. Several open-source ML routines were tested and trained using DMS-MS data in the form of the parent ion's m/z and the compensation voltage required for elution at specific separation voltages between 1500 and 4000 V. The best performing ML model, random forest regression, predicted CCSs with a mean absolute percent error of 2.6 ± 0.4% for analytes excluded from the training set (i.e., out-of-the-bag external validation). This accuracy approaches the inherent statistical error of ∼2.2% for the MobCal-MPI CCS calculations employed for training purposes and the <2% threshold for matching literature CCSs with those obtained on a TWIMS platform.

Corona discharge ion mobility spectrometry combined by homogenizer assisted dispersive liquid-phase microextraction; A rapid and sensitive method for quantification of nortriptyline

In this research, homogenizer assisted dispersive liquid-phase microextraction has been used as an efficient and fast extraction-preconcentration procedure of nortriptyline from different samples. The ultimate analysis of nortriptyline was done by the ion mobility spectrometry technique. A laboratory homogenizer has been employed as the dispersing element for the emulsification of extracting organic solvent. The effective parameters on the extraction process including type and volume of extraction solvent, sample ionic strength and sample solution pH, homogenization time, and dispersing rate were investigated and optimized. The evaluated optimal values of these parameters were found to be 40 µL of dibromomethane as the extraction solvent, sample solutions pH of 10, 10 w/v% of NaCl for adjusting the ionic strength, dispersing rate of 16000 rpm and the homogenizing time of 6 s. The calibration curve was linear in the range of 5 to 200 μg L−1 with the detection limit of 1.9 μg L−1, and the calculated intra- and inter-day precisions based on the corresponding relative standard deviations were found to be 4.0% and 4.6%, respectively. The proposed method was evaluated for the determination of nortriptyline in human breast milk, urine, and a river water samples. The relative recoveries for studied samples were between 91.2 and 105.7%.

Atmospheric Pressure Chemical Ionisation study of selected Volatile Organic Compounds (VOCs) by Ion Mobility Spectrometry coupled with orthogonal acceleration Time Of Flight Mass Spectrometry

In this work, we present an Atmospheric Pressure Chemical Ionisation (APCI) study of several organic solvents relevant to Thin Layer Chromatography (TLC) (methanol, ethanol, propan-2-ol, acetone, ethyl acetate, diethylamine). The solvents were investigated using an Ion Mobility Spectrometry (IMS) combined with orthogonal acceleration Time Of Flight Mass Spectrometry (IMS-oaTOF MS). The solvents were iononised by Reactant Ions (RI – H+.(H2O)3,4 m/z 55 and 73). The ions were analysed using IMS and IMS-oaTOF technique was used to assign the ion mobility peaks to ions according to their m/z. IMS spectra of methanol, ethanol, propan-2-ol and diethylamine exhibited only one peak (reduced ion mobilities of 2.19, 2.07, 1.98, 2.06 cm2s−1V−1 respectively) whereas acetone and ethyl acetate were characterised by two peaks (2.15, 1.85 and 1.94, 1.53 cm2s−1V−1 respectively). The IMS-oaTOF spectrometry assigned IMS peaks of VOCs to M⋅H+(H2O)n. n = 0,1,2 or M2⋅H+ ions, where M represents the solvent molecule.

Ion mobility spectrometry as a fast screening tool for synthetic cannabinoids to uncover drug trafficking in jail via herbal mixtures, paper, food, and cosmetics.

The greatest challenge for European drug policies is how to effectively respond to the dynamic and constantly changing market for new psychoactive substances (NPS). Even small modifications in the chemical structure of substances often allow circumventing existing laws. Also in prison, the consumption of NPS is rising and there is growing evidence that NPS are responsible for a large share of drug-related problems. Ion mobility spectrometry (IMS) is the technique of choice for trace analysis of illicit drugs or explosives at security points, for example airports. Currently, databases of the reduced mobility (K0 ) values are limited to classical drugs and should be completed with data of emerging NPS. In this article, K0 values, LODs (0.7-3.6ng) and drift times of 25 synthetic cannabinoids were evaluated. The data were added to existing databases of IMS which were then applied for fast screening in prison. The detection capability of the portable IMS technique was evaluated by the determination of intra-day (0.089%) and inter-day precision (0.004% to 0.14%), systematic error (0.19%), and separation capability for structurally related NPS. The applicability of the methodology was demonstrated by the successful analysis of 12 different pieces of paper impregnated with synthetic cannabinoids, 7 different cosmetics, and 5 food samples (liquids), spiked with a mixture of narcotic drugs and a synthetic cannabinoid. In addition, 14 herbal mixtures and 36 different casework samples from prisons were analyzed provided by the State Office of Criminal Investigation Rhineland-Palatinate (Germany).

Ambient ionisation mass spectrometry for in situ analysis of intact proteins.

Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein‐protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed.

Simultaneous Improvement of Resolving Power and Signal-to-Noise Ratio Using a Modified Hadamard Transform-Inverse Ion Mobility Spectrometry Technique.

In order to improve the resolving power (RP) and signal-to-noise ratio (SNR) of ion mobility spectrometry (IMS) simultaneously, a modified Hadamard transform-inverse ion mobility spectrometry (MHT-IIMS) technique was developed. In this novel technique, a series of isolating codes were appended to each element of the pseudo random binary sequence (PRBS), and then the modified modulation sequence was formed and used to control the ion gate of the inverse IMS (IIMS). Experimental results demonstrate that the MHT-IIMS technique can significantly enhance the resolving power and SNR simultaneously by measuring the spectra of reaction ions. Furthermore, the gas sample CCl4 and CHCl3 were measured for evaluating the capability of detecting those samples which have single and multiple product ions. The results show that this novel technique is able to simultaneously improve the resolving power and SNR notablely for the real sample detection without any significant instrumental changes. Graphical Abstract ᅟ.

Determination of Histamine and Tyramine in Canned Fish Samples by Headspace Solid-Phase Microextraction Based on a Nanostructured Polypyrrole Fiber Followed by Ion Mobility Spectrometry

This paper describes the development of a simple, rapid, and selective method based on headspace solid-phase microextraction (HS-SPME) combined with ion mobility spectrometry (IMS) for the simultaneous determination of histamine (HIS) and tyramine (TYR) in the canned fish samples. The spectra interferences were eliminated by using a new alternate reagent gas and resulted in an increased sensitivity and selectivity of IMS technique. A dodecylbenzenesulfonate-doped polypyrrole coating was used as a fiber for HS-SPME method. The calibration curves were linear in the range of 30–300 ng g−1 (R2 > 0.994). Limits of detection for HIS and TYR were 3 and 4 ng g−1, respectively. The proposed method was successfully applied to determine HIS and TYR in different canned fish samples without derivatization steps. Method validation was conducted by comparing our results with those obtained through gas chromatography-mass spectrometry method.

A structural examination and collision cross section database for over 500 metabolites and xenobiotics using drift tube ion mobility spectrometry.

The confident identification of metabolites and xenobiotics in biological and environmental studies is an analytical challenge due to their immense dynamic range, vast chemical space and structural diversity. Ion mobility spectrometry (IMS) is widely used for small molecule analyses since it can separate isomeric species and be easily coupled with front end separations and mass spectrometry for multidimensional characterizations. However, to date IMS metabolomic and exposomic studies have been limited by an inadequate number of accurate collision cross section (CCS) values for small molecules, causing features to be detected but not confidently identified. In this work, we utilized drift tube IMS (DTIMS) to directly measure CCS values for over 500 small molecules including primary metabolites, secondary metabolites and xenobiotics. Since DTIMS measurements do not need calibrant ions or calibration like some other IMS techniques, they avoid calibration errors which can cause problems in distinguishing structurally similar molecules. All measurements were performed in triplicate in both positive and negative polarities with nitrogen gas and seven different electric fields, so that relative standard deviations (RSD) could be assessed for each molecule and structural differences studied. The primary metabolites analyzed to date have come from key metabolism pathways such as glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle, while the secondary metabolites consisted of classes such as terpenes and flavonoids, and the xenobiotics represented a range of molecules from antibiotics to polycyclic aromatic hydrocarbons. Different CCS trends were observed for several of the diverse small molecule classes and when urine features were matched to the database, the addition of the IMS dimension greatly reduced the possible number of candidate molecules. This CCS database and structural information are freely available for download at http://panomics.pnnl.gov/metabolites/ with new molecules being added frequently.

Rapid monitoring and sensitive determination of DDT and its metabolites in water sample using solid-phase extraction followed by ion mobility spectrometry

Dichlorodiphenyl trichloroethane (DDT) as an organochlorine compound has been globally used as a pesticide for controlling soil-dwelling insects and treating diseases such as malaria and typhus. The degradation products of DDT and its metabolites have also negative effects on the environment. The present study has investigated the determination of DDT and its metabolites in water sample using ion mobility spectrometry (IMS) as a rapid and sensitive detection technique. For this purpose, DDT and its metabolites were extracted using reverse phase solid-phase extraction (SPE) from water samples. The samples were then recovered by eluting with methanol and finally, quantified using the corona discharge IMS technique. Injection and oven temperatures and the effect of dopant were optimized as experimental parameters influencing both detection and determination efficiencies. Degradation of DDT in IMS drift tube was studied and reduced mobility values of DDT and its metabolites were calculated. The developed method was validated using water sample to obtain good results for the determination of DDT at low levels (1 ng ml−1) while spiked recoveries were obtained to be between 95.0–96.7%. The proposed method based on IMS proved to be a simple, inexpensive, rapid and sensitive procedure for the fast monitoring and determination of DDT and its main metabolites in water sample.

Opposing Effects of Cucurbit[7]uril and 1,2,3,4,6-Penta-O-galloyl-β-d-glucopyranose on Amyloid β25-35 Assembly.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by extracellular deposits of amyloid β protein (Aβ) in the brain. The conversion of soluble monomers to amyloid Aβ fibrils is a complicated process and involves several transient oligomeric species, which are widely believed to be highly toxic and play a crucial role in the etiology of AD. The development of inhibitors to prevent formation of small and midsized oligomers is a promising strategy for AD treatment. In this work, we employ ion mobility spectrometry (IMS), transmission electron microscopy (TEM), and molecular dynamics (MD) simulations to elucidate the structural modulation promoted by two potential inhibitors of Aβ oligomerization, cucurbit[7]uril (CB[7]) and 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose (PGG), on early oligomer and fibril formation of the Aβ25-35 fragment. One and two CB[7] molecules bind to Aβ25-35 monomers and dimers, respectively, and suppress aggregation by remodeling early oligomer structures and inhibiting the formation of higher-order oligomers. On the other hand, nonselective binding was observed between PGG and Aβ25-35. The interactions between PGG and Aβ25-35, surprisingly, enhanced the formation of Aβ aggregates by promoting extended Aβ25-35 conformations in both homo- and hetero-oligomers. When both ligands were present, the inhibitory effect of CB[7] overrode the stimulatory effect of PGG on Aβ25-35 aggregation, suppressing the formation of large amyloid oligomers and eliminating the structural conversion from isotropic to β-rich topologies induced by PGG. Our results provide mechanistic insights into CB[7] and PGG action on Aβ oligomerization. They also demonstrate the power of the IMS technique to investigate mechanisms of multiple small-molecule agents on the amyloid formation process.

High Resolution Trapped Ion Mobility Spectrometery of Peptides

In the present work, we employ trapped ion mobility spectrometry (TIMS) for conformational analysis of several model peptides. The TIMS distributions are extensively compared to recent ion mobility spectrometry (IMS) studies reported in the literature. At a resolving power (R) exceeding 250, many new features, otherwise hidden by lower resolution IMS analyzers, are revealed. Though still principally limited by the plurality of conformational states, at present, TIMS offers R up to ∼3 to 8 times greater than modern drift tube or traveling wave IMS techniques, respectively. Unlike differential IMS, TIMS not only is able to resolve congested conformational features but also can be used to determine information about their relative size, via the ion-neutral collision cross section, offering a powerful new platform to probe the structure and dynamics of biochemical systems in the gas phase.

Carbohydrate and Glycoconjugate Analysis by Ion Mobility Mass Spectrometry: Opportunities and Challenges

Carbohydrates are characterized by a truly remarkable level of structural diversity which stands in accord with a corresponding functional diversity arguably unsurpassed by any other class of biomolecule. As metabolites, structural components, mediators of molecular interaction, and modifiers of other biomolecules, carbohydrates play central roles in numerous areas of biochemistry. Detailed molecular characterization of these classes of compounds poses a number of significant analytical challenges, a number of which arise from carbohydrate isomerism combined with the ability of monosaccharides to serve as the basis for branched oligosaccharide structures. Ion mobility spectrometry (IMS) represents a particularly appealing analytical methodology for addressing such hurdles. Because IMS provides for separation of gasphase ions according to their “shape to charge ratios,” this approach is well-suited to provide for the recognition, distinction, and resolution of isomeric carbohydrates and glycoconjugates. IMS is also readily coupled to well-established mass spectrometry ionization methods and analyzers, which provide complementary information on mass to charge ratios and allow tandem mass spectrometry dissociation experiments which can supply further structural information. Here, we offer a brief introduction to carbohydrate structure and diversity (including monosaccharides, oligosaccharides, and glycoconjugates thereof), followed by an overview of relevant IMS techniques (drift tube ion mobility spectrometry; high-field asymmetric waveform ion mobility spectrometry; and traveling wave ion mobility spectrometry). We then provide an overview of the literature on IMS as applied to the study of free carbohydrates (including isomer separation and general structural characterization) and glycoconjugates (including released glycans and intact glycoconjugates). Keywords: Carbohydrates, glycoconjugates, glycopeptides, glycoproteins, ion mobility spectrometry, mass spectrometry, oligosaccharides.

Site-Specific Characterization of d-Amino Acid Containing Peptide Epimers by Ion Mobility Spectrometry

Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues.

Ion mobility spectrometry‐mass spectrometry (IMS‐MS) of small molecules: Separating and assigning structures to ions

The phenomenon of ion mobility (IM), the movement/transport of charged particles under the influence of an electric field, was first observed in the early 20th Century and harnessed later in ion mobility spectrometry (IMS). There have been rapid advances in instrumental design, experimental methods, and theory together with contributions from computational chemistry and gas-phase ion chemistry, which have diversified the range of potential applications of contemporary IMS techniques. Whilst IMS-mass spectrometry (IMS-MS) has recently been recognized for having significant research/applied industrial potential and encompasses multi-/cross-disciplinary areas of science, the applications and impact from decades of research are only now beginning to be utilized for "small molecule" species. This review focuses on the application of IMS-MS to "small molecule" species typically used in drug discovery (100-500 Da) including an assessment of the limitations and possibilities of the technique. Potential future developments in instrumental design, experimental methods, and applications are addressed. The typical application of IMS-MS in relation to small molecules has been to separate species in fairly uniform molecular classes such as mixture analysis, including metabolites. Separation of similar species has historically been challenging using IMS as the resolving power, R, has been low (3-100) and the differences in collision cross-sections that could be measured have been relatively small, so instrument and method development has often focused on increasing resolving power. However, IMS-MS has a range of other potential applications that are examined in this review where it displays unique advantages, including: determination of small molecule structure from drift time, "small molecule" separation in achiral and chiral mixtures, improvement in selectivity, identification of carbohydrate isomers, metabonomics, and for understanding the size and shape of small molecules. This review provides a broad but selective overview of current literature, concentrating on IMS-MS, not solely IMS, and small molecule applications.

Real-time monitoring of breath ammonia during haemodialysis: use of ion mobility spectrometry (IMS) and cavity ring-down spectroscopy (CRDS) techniques

The diffusion of high-performance analytical technology has opened prospects for breath diagnosis as a non-invasive diagnostic tool. In this study, ion mobility spectrometry (IMS) and cavity ring-down spectroscopy (CRDS) techniques were used to analyse ammonia gas (NH3) in real-time in breath from patients undergoing haemodialysis (HD) treatment and any correlation with blood urea nitrogen (BUN) levels and Kt/V were investigated. We studied 20 patients on intermittent HD treatment. The first breath samples were taken before the start of dialysis and further breath samples were taken every hour during the treatment and after the end of the session. An evaluation was also made of 20 healthy volunteers, acting as controls [healthy subjects (HS)]. Breath ammonia concentrations were higher in CRDS-HD (914.5±301.4 versus 280±120 parts per billion (p.p.b.), P<0.0001) and IMS-HD patients (964.4±402.4 versus 280±120 p.p.b., P<0.0001) than in HS. We assessed real-time variations in the levels of NH(3) and showed a continuous decrease in the levels of NH3. Expired NH3 correlated directly with BUN levels, both in the IMS-HD (P=0.002; r=0.84; P=0.009; r=0.76) and in the CRDS-HD group (P=0.005; r=0.80; P=0.008; r=0.77), respectively, both before and at the end of dialysis. A direct correlation with Kt/V was found in both groups studied (IMS-HD: P=0.003; r=0.82; CRDS-HD: P=0.006; r=0.79). Breath monitoring of NH3 with IMS and CRDS techniques could be useful to assess the real-time clinical status of patients during HD. By using pre-dialysis ammonia values, an approximate calculation of the Kt/Vurea ratio can be established.

Differentiation of Compact and Extended Conformations of Di-Ubiquitin Conjugates with Lysine-Specific Isopeptide Linkages by Ion Mobility-Mass Spectrometry

Modification of ubiquitin, a key cellular regulatory polypeptide of 76 amino acids, to polyubiquitin conjugates by lysine-specific isopeptide linkage at one of its seven lysine residues has been recognized as a central pathway determining its biochemical properties and cellular functions. Structural details and differences of distinct lysine-isopeptidyl ubiquitin conjugates that reflect their different functions and reactivities, however, are only partially understood. Ion mobility spectrometry (IMS) combined with mass spectrometry (MS) has recently emerged as a powerful tool for probing conformations and topology involved in protein interactions by an electric field-driven separation of polypeptide ions through a drift gas. Here we report the conformational characterization and differentiation of Lys63- and Lys48-linked ubiquitin conjugates by IMS-MS. Lys63- and Lys48-linked di-ubiquitin conjugates were prepared by recombinant bacterial expression and by chemical synthesis using a specific chemical ligation strategy, and characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry, circular dichroism spectroscopy, and molecular modeling. IMS-MS was found to be an effective tool for the identification of structural differences of ubiquitin complexes in the gas phase. The comparison of collision cross-sections of Lys63- and Lys48-linked di-ubiquitin conjugates showed a more elongated conformation of Lys63-linked di-ubiquitin. In contrast, the Lys48-linked di-ubiquitin conjugate showed a more compact conformation. The IMS-MS results are consistent with published structural data and a comparative molecular modeling study of the Lys63- and Lys48-linked conjugates. The results presented here suggest IMS techniques can provide information that complements MS measurements in differentiating higher-order polyubiquitins and other isomeric protein linkages.

Specific O2− generation in corona discharge for ion mobility spectrometry

This study deals with O2− generation in corona discharge (CD) in point to plane geometry for single flow ion mobility spectrometry (IMS) with gas outlet located behind the ionization source. We have designed CD of special geometry in order to achieve the high O2− yield. Using this ion source we have achieved in zero air conditions that up to 74% all negative ions were O2− or O2−(H2O). It has been demonstrated that the non-electronegative nitrogen positively influences the efficiency of O2− generation in O2/N2 mixtures. The reduced ion mobility of 2.27cm2V−1s−1 has been measured for O2−/O2−(H2O) ions in zero air. Additional ions detected in zero air (less than 200ppb CO2) using the mass spectrometric and IMS technique were, NO2−, N2O2− (2.37cm2V−1s−1), NO3−, N2O3− and N2O3−(H2O). The CO3− and CO4− ions have been detected after the introduction of 5ppm CO2 into zero air.