Examining DNA Structures with In-droplet Hydrogen/Deuterium Exchange Mass Spectrometry.

Capillary vibrating sharp-edge spray ionization (cVSSI) combined with hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been utilized to characterize different solution-phase DNA conformers including DNA G-quadruplex topologies as well as triplex DNA and duplex DNA. In general, G-quadruplex DNA shows a wide range of protection of hydrogens extending from ~12% to ~21% deuterium incorporation. Additionally, the DNA sequences selected to represent parallel, antiparallel, and hybrid G-quadruplex topologies exhibit slight differences in deuterium uptake levels which appear to loosely relate to overall conformer stability. Notably, the exchange level for one of the hybrid sequence sub topologies of G-quadruplex DNA (24 TTG) is significantly different (compared with the others studied here) despite the DNA sequences being highly comparable. For the quadruplex-forming sequences, correlation analysis suggests protection of base hydrogens involved in tetrad hydrogen bonding. For duplex DNA ~19% deuterium incorporation is observed while only ~16% is observed for triplex DNA. This increased protection of hydrogens may be due to the added backbone scaffolding and Hoogsteen base pairing of the latter species. These experiments lay the groundwork for future studies aimed at determining the structural source of this protection as well as the applicability of the approach for ascertaining different oligonucleotide folds, co-existing conformations, and/or overall conformer flexibility.

In-droplet hydrogen-deuterium exchange to examine protein/peptide solution conformer heterogeneity.

RATIONALE: Many different structure analysis techniques are not capable of probing the heterogeneity of solution conformations. Here, we examine the ability of in-droplet hydrogen-deuterium exchange (HDX) to directly probe solution conformer heterogeneity of a protein with mass spectrometry (MS) detection. METHODS: Two vibrating capillary vibrating sharp-edge spray ionization (cVSSI) devices have been arranged such that they generate microdroplet plumes of the analyte and D2 O reagent, which coalesce to form reaction droplets where HDX takes place in the solution environment. The native HDX-MS setup has been first explored for two model peptides that have distinct structural compositions in solution. The effectiveness of the multidevice cVSSI-HDX in illustrating structural details has been further exploited to investigate coexisting solution-phase conformations of the protein ubiquitin. RESULTS: In-droplet HDX reveals decreased backbone exchange for a model peptide that has a greater helix-forming propensity. Differences in intrinsic rates of the alanine and serine residues may account for much of the observed protection. The data allow the first estimates of backbone exchange rates for peptides undergoing in-droplet HDX. That said, the approach may hold greater potential for investigating the tertiary structure and structural transitions of proteins. For ubiquitin protein, HDX reactivity differences suggest that multiple conformers are present in native solutions. The addition of methanol to buffered aqueous solutions of ubiquitin results in increased populations of solution conformers of higher reactivity. Data analysis suggests that partially folded conformers such as the A-state of ubiquitin increase with methanol content; the native state may be preserved to a limited degree even under stronger denaturation conditions. CONCLUSION: The deuterium uptake after in-droplet HDX has been observed to correspond to some degree with peptide backbone hydrogen protection based on differences in intrinsic rates of exchange. The presence of coexisting protein solution structures under native and denaturing solution conditions has been distinguished by the isotopic distributions of deuterated ubiquitin ions.

Portable mass spectrometry system: instrumentation, applications, and path to 'omics analysis.

Mass spectrometry (MS) is an information rich analytical technique and plays a key role in various 'omics studies. Standard mass spectrometers are bulky and operate at high vacuum, which hinder their adoption by the broader community and utility in field applications. Developing portable mass spectrometers can significantly expand the application scope and user groups of MS analysis. This review discusses the basics and recent advancements in the development of key components of portable mass spectrometers including ionization source, mass analyzer, detector, and vacuum system. Further, major areas where portable mass spectrometers are applied are also discussed. Finally, a perspective on the further development of portable mass spectrometers including the potential benefits for 'omics analysis is provided.

Oxidation Promotes Distinct Huntingtin Aggregates in the Presence and Absence of Membranes.

Expansion of a polyglutamine (polyQ) domain within the first exon of the huntingtin (htt) protein is the underlying cause of Huntington's disease, a genetic neurodegenerative disorder. PolyQ expansion triggers htt aggregation into oligomers, fibrils, and inclusions. The 17 N-terminal amino acids (Nt17) of htt-exon1, which directly precede the polyQ domain enhances polyQ fibrillization and functions as a lipid-binding domain. A variety of post-translational modifications occur within Nt17, including oxidation of two methionine residues. Here, the impact of oxidation within Nt17 on htt aggregation both in the presence and absence of lipid membranes was investigated. Treatment with hydrogen peroxide (H2O2) reduced fibril formation in a dose-dependent manner, resulting in shorter fibrils and an increased oligomer population. With excessive H2O2 treatments, fibrils developed a unique morphological feature around their periphery. In the presence of total brain lipid vesicles, H2O2 impacted fibrillization in a similar manner. That is, oligomerization was promoted at the expense of fibril elongation. The interaction of unoxidized and oxidized htt with supported lipid bilayers was directly observed using in situ atomic force microscopy. Without oxidation, granular htt aggregates developed on the bilayer surface. However, in the presence of H2O2, distinct plateau-like regions initially developed on the bilayer surface that gave way to rougher patches containing granular aggregates. Collectively, these observations suggest that oxidation of methionine residues within Nt17 plays a crucial role in both the aggregation of htt and its ability to interact with lipid surfaces.

Development of cVSSI-APCI for the Improvement of Ion Suppression and Matrix Effects in Complex Mixtures.

The new ionization technique termed vibrating sharp-edge spray ionization (cVSSI) has been coupled with corona discharge to investigate atmospheric pressure chemical ionization (APCI) capabilities. The optimized source was evaluated for its ability to enhance ion signal intensity, overcome matrix effects, and limit ion suppression. The results have been compared with state-of-the-art ESI source performance as well as a new APCI-like source. In methanol, the ion signal intensity increased 10-fold and >10-fold for cocaine and the suppressed analytes, respectively. The ability to overcome ion suppression was improved from 2-fold to 16-fold for theophylline and vitamin D2, respectively. For aqueous samples, ion signal levels increased by two orders of magnitude for all analytes. In both solvent systems, the signal-to-noise ratios also increased for all suppressed analytes. One example of the characterization of low-ionizing (by ESI or cVSSI alone) species in the presence of high-ionizing species by direct analysis from a cotton swab is presented. The work is discussed with respect to the advantages of cVSSI-APCI for direct, in situ, and field analyses.

Nanoflow Sheath Voltage-Free Interfacing of Capillary Electrophoresis and Mass Spectrometry for the Detection of Small Molecules.

Coupling capillary electrophoresis (CE) to mass spectrometry (MS) is a powerful strategy to leverage a high separation efficiency with structural identification. Traditional CE-MS interfacing relies upon voltage to drive this process. Additionally, sheathless interfacing requires that the electrophoresis generates a sufficient volumetric flow to sustain the ionization process. Vibrating sharp-edge spray ionization (VSSI) is a new method to interface capillary electrophoresis to mass analyzers. In contrast to traditional interfacing, VSSI is voltage-free, making it straightforward for CE and MS. New nanoflow sheath CE-VSSI-MS is introduced in this work to reduce the reliance on the separation flow rate to facilitate the transfer of analyte to the MS. The nanoflow sheath VSSI spray ionization functions from 400 to 900 nL/min. Using the new nanoflow sheath reported here, volumetric flow rate through the separation capillary is less critical, allowing the use of a small (i.e., 20 to 25 µm) inner diameter separation capillary and enabling the use of higher separation voltages and faster analysis. Moreover, the use of a nanoflow sheath enables greater flexibility in the separation conditions. The nanoflow sheath is operated using aqueous solutions in the background electrolyte and in the sheath, demonstrating the separation can be performed under normal and reversed polarity in the presence or absence of electroosmotic flow. This includes the use of a wider pH range as well. The versatility of nanoflow sheath CE-VSSI-MS is demonstrated by separating cationic, anionic, and zwitterionic molecules under a variety of separation conditions. The detection sensitivity observed with nanoflow sheath CE-VSSI-MS is comparable to that obtained with sheathless CE-VSSI-MS as well as CE-MS separations with electrospray ionization interfacing. A bare fused silica capillary is used to separate cationic ß-blockers with a near-neutral background electrolyte at concentrations ranging from 1.0 nM to 1.0 µM. Under acidic conditions, 13 amino acids are separated with normal polarity at a concentration ranging from 0.25 to 5 µM. Finally, separations of anionic compounds are demonstrated using reversed polarity under conditions of suppressed electroosmotic flow through the use of a semipermanent surface coating. With a near-neutral separation electrolyte, anionic nonsteroidal anti-inflammatory drugs are detected over a concentration range of 0.1 to 5.0 µM.

Rapid and flexible online desalting using Nafion-coated melamine sponge for mass spectrometry analysis.

RATIONALE: The performance of mass spectrometry (MS) analysis is often affected by the presence of salt ions. To achieve optimal MS detection results, desalting is necessary for samples with high salt concentrations. We report a rapid, low-cost and flexible online desalting method using Nafion-coated sponge. This method is easy to perform and can be implemented to a wide range of customized fluidic systems. METHODS: Nafion-coated melamine sponge was fabricated by soaking a glass tube containing a melamine sponge in Nafion solution and then drying overnight. The online desalting workflow is comprised of three major parts: (1) Syringe pump, which provides a continuous flow for the online fluid system; (2) Nafion sponge in a glass tube, where the online desalting of sample solution happens; (3) Capillary Vibrating Sharp-Edge Spray Ionization (cVSSI), which is an ionization technique to ionize the desalted analytes. RESULTS: Effective online desalting of a 10 mM NaCl solution was demonstrated for a wide range of molecules including small molecules, peptides, DNAs, and proteins using a flow rate of 10 µL/min. By incorporating multiple pieces of the Nafion-coated sponge, effective desalting for ubiquitin and cytochrome c (Cyt-c) from physiological buffers, including phosphate-buffered saline (PBS) and tris-buffered saline (TBS), were also achieved. For molecules that are sensitive to low pH conditions after desalting, a R-SO3 NH4 -type Nafion-coated sponge was fabricated. Desalting of ubiquitin, oligosaccharide, and DNA oligomers from 10 mM NaCl or 10 mM KCl solutions was demonstrated. CONCLUSIONS: Flexible, low-cost, and efficient online desalting was achieved by the Nafion-coated sponge. A variety of molecules ranging from small molecules, peptides, proteins to oligosaccharides and DNAs can be desalted for MS analysis. The desalting by Nafion sponge has great potential for desalting applications that require customized fluidic design and rapid analysis.

Integrated sample desalting, enrichment, and ionization on an omniphobic glass slide for direct mass spectrometry analysis.

RATIONALE: Direct and rapid mass spectrometry (MS) analysis is desired for many applications including environmental monitoring, forensic analysis, chemical and biological defense, and point-of-care testing. However, sample pretreatment is often necessary for analyzing targets from complex matrices using MS due to ion suppression. To achieve rapid MS analysis calls for simple and efficient solutions for sample processing and ionization. Here, a simple sample pretreatment and ionization workflow is reported, which achieves sample desalting, enrichment, and ionization on a single glass slide. METHODS: Desalting is achieved based on crystallization and re-dissolution-induced spontaneous separation of analytes and salt. Efficient sample enrichment is achieved during the crystallization process by modifying the glass surface with an omniphobic coating. Finally, vibrating sharp-edge spray ionization is employed to ionize the target molecules directly on the glass slide. Thus, all the necessary sample operations prior to MS analysis are completed on the sample glass slide. RESULTS: Efficient sample enrichment on the omniphobic glass slide is first visualized using food dyes. The benefits of the desalting and enrichment steps for detecting macrolide antibiotics in 1× phosphate buffered saline (PBS) solutions are demonstrated by comparing samples with different treatment procedures. Finally, quantification of macrolide antibiotics from PBS and serum samples is demonstrated. A linear range between 2 nM and 10 µM has been achieved for the serum sample with a limit of detection of 1 nM. CONCLUSIONS: A simple, flexible, low-cost, and highly integrated workflow for detecting target molecules from complex matrices using MS is demonstrated. This method will be valuable to many applications that require rapid and efficient MS analysis of complex samples.

Vibrating Sharp-edge Spray Ionization (VSSI) for voltage-free direct analysis of samples using mass spectrometry.

RATIONALE: The development of miniaturized and field portable mass spectrometers could not succeed without a simple, compact, and robust ionization source. Here we present a voltage-free ionization method, Vibrating Sharp-edge Spray Ionization (VSSI), which can generate a spray of liquid samples using only one standard microscope glass slide to which a piezoelectric transducer is attached. Compared with existing ambient ionization methods, VSSI eliminates the need for a high electric field (~5000 V·cm-1 ) for spray generation, while sharing a similar level of simplicity and flexibility with the simplest direct ionization techniques currently available such as paper spray ionization (PSI) and other solid substrate-based electrospray ionization methods. METHODS: The VSSI device was fabricated by attaching a piezoelectric transducer onto a standard glass microscope slide using epoxy glue. Liquid sample was aerosolized by either placing a droplet onto the vibrating edge of the glass slide or touching a wet surface with the glass edge. Mass spectrometric detection was achieved by placing the VSSI device 0.5-1 cm from the inlet of the mass spectrometer (Q-Exactive, ThermoScientific). RESULTS: VSSI is demonstrated to ionize a diverse array of chemical species, including small organic molecules, carbohydrates, peptides, proteins, and nucleic acids. Preliminary sensitivity experiments show that high-quality mass spectra of acetaminophen can be obtained by consuming 100 femtomoles of the target. The dual spray of VSSI was also demonstrated by performing in-droplet denaturation of ubiquitin. Finally, due to the voltage-free nature and the direct-contact working mode of VSSI, it has been successfully applied for the detection of chemicals directly from human fingertips. CONCLUSIONS: Overall, we report a compact ionization method based on vibrating sharp-edges. The simplicity and voltage-free nature of VSSI make it an attractive option for field portable applications or analyzing biological samples that are sensitive to high voltage or difficult to access by conventional ionization methods.

Nucleation Inhibition of Huntingtin Protein (htt) by Polyproline PPII Helices: A Potential Interaction with the N-Terminal α-Helical Region of Htt.

Huntington's disease is a genetic neurodegenerative disorder characterized by the formation of amyloid fibrils of the huntingtin protein (htt). The 17-residue N-terminal region of htt (Nt17) has been implicated in the formation of early phase oligomeric species, which may be neurotoxic. Because tertiary interactions with a downstream (C-terminal) polyproline (polyP) region of htt may disrupt the formation of oligomers, which are precursors to fibrillar species, the effect of co-incubation of a region of htt with a 10-residue polyP peptide on oligomerization and fibrillization has been examined by atomic force microscopy. From multiple, time-course experiments, morphological changes in oligomeric species are observed for the protein/peptide mixture and compared with the protein alone. Additionally, an overall decrease in fibril formation is observed for the heterogeneous mixture. To consider potential sites of interaction between the Nt17 region and polyP, mixtures containing Nt17 and polyP peptides have been examined by ion mobility spectrometry and gas-phase hydrogen-deuterium exchange coupled with mass spectrometry. These data combined with molecular dynamics simulations suggest that the C-terminal region of Nt17 may be a primary point of contact. One interpretation of the results is that polyP may possibly regulate Nt17 by inducing a random coil region in the C-terminal portion of Nt17, thus decreasing the propensity to form the reactive amphipathic α-helix. A separate interpretation is that the residues important for helix-helix interactions are blocked by polyP association.

Facile Improvement of Negative Ion Mode Electrospray Ionization Using Capillary Vibrating Sharp-Edge Spray Ionization.

Electrospray ionization (ESI) is often affected by corona discharge when spraying 100% aqueous solutions as the voltage that induces discharge can be well below the onset voltage of ESI. As a result, it is especially challenging to perform native mass spectrometry in negative ion mode where 100% aqueous solution is preferred. Here we report a simple instrumentation method to improve the performance of ESI in negative ion mode based on capillary vibrating sharp-edge spray ionization. By attaching a fused silica capillary emitter to a vibrating glass slide, improved signal quality is achieved for various analytes in aqueous solutions over applying ESI alone. Compared to commercial ESI sources using nebulization gas to reduce discharge, 10-100-fold enhancement in signal intensity and 3-10-fold improvement in S/N are achieved for various kinds of molecules including DNA, peptides, proteins, and oligosaccharides. Finally, the new method demonstrates utility for native mass spectrometry analysis of proteins and G-quadruplex DNA. The present method is expected to have great potential to be adopted by the scientific community because of its improved analytical performance, simplicity, and low cost.

Recommendations for reporting ion mobility Mass Spectrometry measurements.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

Hydrogen Peroxide Modifies Aß-Membrane Interactions with Implications for Aß40 Aggregation.

Alzheimer's disease (AD) is pathologically characterized by the formation of extracellular senile plaques, predominately comprised of aggregated ß-amyloid (Aß), deposited in the brain. Aß aggregation can result in a myriad of distinct aggregate species, from soluble oligomers to insoluble fibrils. Aß strongly interacts with membranes, which can be linked to a variety of potential toxic mechanisms associated with AD. Oxidative damage accompanies the formation of Aß aggregates, with a 10-50% proportion of Aß aggregates being oxidized in vivo. Hydrogen peroxide (H2O2) is a reactive oxygen species implicated in a number of neurodegenerative diseases. Recent evidence has demonstrated that the H2O2 concentration fluctuates rapidly in the brain, resulting in large concentration spikes, especially in the synaptic cleft. Here, the impact of environmental H2O2 on Aß aggregation in the presence and absence of lipid membranes is investigated. Aß40 was exposed to H2O2, resulting in the selective oxidation of methionine 35 (Met35) to produce Aß40Met35[O]. While oxidation mildly reduced the rate of Aß aggregation and produced a distinct fibril morphology at high H2O2 concentrations, H2O2 had a much more pronounced impact on Aß aggregation in the presence of total brain lipid extract vesicles. The impact of H2O2 on Aß aggregation in the presence of lipids was associated with a reduced affinity of Aß for the vesicle surface. However, this reduced vesicle affinity was predominately associated with lipid peroxidation rather than Aß oxidation.

Ion Mobility, Hydrogen/Deuterium Exchange, and Isotope Scrambling: Tools to Aid Compound Identification in 'Omics Mixtures.

Liquid chromatography tandem mass spectrometry (LC-MS/MS), a widely used method for comparative 'omics analysis, experiences challenges with compound identification due to matrix effects, difficulty in separating isomer and isobaric ions, and long analysis times. Ion mobility spectrometry (IMS) has proven to be useful in separating isomer and isobar ions; however, IMS-MS suffers from decreased peak capacity due to the correlation in ion size and mass. In proof-of-principle experiments, the use of gas-phase hydrogen/deuterium exchange (HDX) combined with IMS-MS/MS techniques is demonstrated to offer advantages for compound identification. Measurements providing unique information for ions include m/z value, drift time in He buffer gas, drift time in He and D2O buffer gases, deuterium incorporation pattern (isotopic distribution), deuterium incorporation pattern after collisional activation, and fragment ion deuterium incorporation pattern upon collision-induced dissociation (CID). These techniques are here shown to be highly reproducible (drift time coefficients of variation < 1.0% and isotopic pattern root-mean-square deviations of 0.5-1.5%) while demonstrating an increased ability to distinguish individual molecules from diverse classes of compounds (peptides, catecholamines, nucleosides, amino acids, etc.). The concept of using such (and similar) information for rapid, high-throughput molecular identification via database searching of standard libraries is briefly discussed, and an example of such usage is presented for a bonafide metabolite extract sample.

Huntingtin N-Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues.

Early stage oligomer formation of the huntingtin protein may be driven by self-association of the 17-residue amphipathic α-helix at the protein's N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington's disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation. Native electrospray ion mobility spectrometry-mass spectrometry (IMS-MS) techniques and molecular dynamics simulations (MDS) have been applied to study coexisting monomer and multimer conformations of Nt17, independent of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer ion structures comprise a helix-turn-coil configuration and a helix-extended-coil region. Elongated dimer species comprise partially helical monomers arranged in an antiparallel geometry. This stacked helical bundle may represent the earliest stages of Nt17-driven oligomer formation. Nt17 monomers and multimers have been further probed using diethylpyrocarbonate (DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6 are modified at higher DEPC concentrations, which led to the formation of an intermediate monomer structure. These modifications resulted in decreased extended monomer ion conformers, as well as a reduction in multimer formation. From the MDS experiments for the dimer ions, Lys6 residues in both monomer constituents interact with Ser16 and Glu12 residues on adjacent peptides; therefore, the decrease in multimer formation could result from disruption of these or similar interactions. This work provides a structurally selective model from which to study Nt17 self-association and provides critical insight toward Nt17 multimerization and, possibly, the early stages of huntingtin exon 1 aggregation.

Online deuterium hydrogen exchange and protein digestion coupled with ion mobility spectrometry and tandem mass spectrometry.

Online deuterium hydrogen exchange (DHX) and pepsin digestion (PD) is demonstrated using drift tube ion mobility spectrometry (DTIMS) coupled with linear ion trap (LTQ) mass spectrometry (MS) with electron transfer dissociation (ETD) capabilities. DHX of deuterated ubiquitin, followed by subsequent quenching and digestion, is performed within ∼60 s, yielding 100% peptide sequence coverage. The high reproducibility of the IMS separation allows spectral feature matching between two-dimensional IMS-MS datasets (undeuterated and deuterated) without the need for dataset alignment. Extracted ion drift time distributions (XIDTDs) of deuterated peptic peptides are mobility-matched to corresponding XIDTDs of undeuterated peptic peptides that were identified using collision-induced dissociation (CID). Matching XIDTDs allows a straightforward identification and deuterium retention evaluation for labeled peptides. Aside from the mobility separation, the ion trapping capabilities of the LTQ, combined with ETD, are demonstrated to provide single-residue resolution. Deuterium retention for the c- series ions across residues M(1)-L(15) and N(25)-R(42) are in good agreement with the known secondary structural elements within ubiquitin.

Advances in ion mobility-mass spectrometry instrumentation and techniques for characterizing structural heterogeneity.

Over the last decade, the field of ion mobility-mass spectrometry (IM-MS) has experienced dramatic growth in its application toward ion structure characterization. Enabling advances in instrumentation during this time period include improved conformation resolution and ion sensitivity. Such advances have rendered IM-MS a powerful approach for characterizing samples presenting a diverse array of ion structures. The structural heterogeneity that can be interrogated by IM-MS techniques now ranges from samples containing mixtures of small molecules exhibiting a variety of structural types to those containing very large protein complexes and subcomplexes. In addition to this diversity, IM-MS techniques have been used to probe spontaneous and induced structural transformations occurring in solution or the gas phase. To support these measurement efforts, significant advances have been made in theoretical methods aimed at translating IM-MS data into structural information. These efforts have ranged from providing more reliable trial structures for comparison to the experimental measurements to dramatically reducing the time required to calculate collision cross sections for such structures. In this short review, recent advances in developments in IM-MS instrumentation, techniques, and theory are discussed with regard to their implications for characterization of gas- and solution-phase structural heterogeneity.

A new ion mobility-linear ion trap instrument for complex mixture analysis.

A new instrument that couples a low-pressure drift tube with a linear ion trap mass spectrometer is demonstrated for complex mixture analysis. The combination of the low-pressure separation with the ion trapping capabilities provides several benefits for complex mixture analysis. These include high sensitivity, unique ion fragmentation capabilities, and high reproducibility. Even though the gas-phase separation and the mass measurement steps are each conducted in an ion filtering mode, detection limits for mobility-selected peptide ions are in the tens of attomole range. In addition to ion separation, the low-pressure drift tube can be used as an ion fragmentation cell yielding mobility-resolved fragment ions that can be subsequently analyzed by multistage tandem mass spectrometry (MS(n)) methods in the ion trap. Because of the ion trap configuration, these methods can be comprised of any number (limited by ion signal) of collision-induced dissociation (CID) and electron transfer dissociation (ETD) processes. The high reproducibility of the gas-phase separation allows for comparison of two-dimensional ion mobility spectrometry (IMS)-MS data sets in a pixel-by-pixel fashion without the need for data set alignment. These advantages are presented in model analyses representing mixtures encountered in proteomics and metabolomics experiments.

Structural Insights into Linkage-Specific Ubiquitin Chains Using Ion Mobility Mass Spectrometry.

The structural characterization and differentiation of four types of oligoubiquitin conjugates [linear (Met1)-, Lys11-, Lys48-, Lys63-linked di-, tri-, and tetraubiquitin chains] using ion mobility mass spectrometry are reported. A comparison of collision cross sections for the same linkage of di-, tri-, and tetraubiquitin chains shows differences in conformational elongation for higher charge states due to the interplay of linkage-derived structure and Coulombic repulsion. For di- and triubiquitin chains, this elongation results in a single narrow feature representing an elongated conformation type for multiple higher charge state species. In contrast, higher charge state tetraubiquitin species do not form a single conformer type as readily. A comparison of different linkages in tetraubiquitin chains reveals greater similarity in conformation type at lower charge states; with increasing charge state, the four linkage types diverge in the relative proportions of elongated conformer types with Met1- ≥ Lys11- > Lys63- > Lys48-linkage. These differences in conformational trends could be discussed with respect to biological functions of linkage-specific polyubiquitinated proteins.

Oscillations of chiral preference in proline clusters.

Ion mobility/mass spectrometry techniques are used to study the chiral preferences of small proline clusters (containing 2 to 23 proline monomers) produced by electrospray ionization. By varying the composition of the electrospray solution from enantiomerically pure (100% L or 100% D) to racemic (50:50 L:D), it is possible to delineate which cluster sizes prefer homochiral (resolved) or heterochiral (antiresolved) compositions. The results show a remarkable oscillation in chiral preference. Singly protonated clusters, [xPro+H](+) (where x corresponds to the number of prolines), favor homochiral assemblies (for x = 4, 6, 11 and 12); heterochiral structures are preferred (although the preferences are not as strong) for x = 5 and 7. Larger, doubly protonated clusters [xPro+2H](2+) favor homochiral assemblies for x = 18, 19, and 23 and heterochiral structures for x = 14, 16, 17, 20, 21, and 22. Some of the variations that are observed can be rationalized through simple structures that would lead to especially stable geometries. It is suggested that some antiresolved clusters, such as [22Pro+2H](2+), may be comprised of resolved D- and L-proline domains.