U-Shaped Mobility Analyzer: A Compact and High-Resolution Counter-Flow Ion Mobility Spectrometer.

In recent years, there has been a rapid increase in the use of counter-flow-type ion mobility spectrometers (IMS) due to their improved resolution and functionality. In this study, we developed a new type of counter-flow ion mobility device named the U-shaped mobility analyzer (UMA) for coupling with a mass spectrometer, where the analyte ions could travel along a gas flow in the first channel of the UMA device and then against a gas flow in the second channel of the device. Hence, a mobility band-pass filter was formed by setting different electric fields in the two channels, which enables high-resolution mobility selection of analyte ions. A resolution of ca. 180 was achieved for singly charged small organic molecules, and a resolution of up to ca. 370 was achieved for multiply charged +15 myoglobin. It was thus demonstrated that this filtering function can greatly enhance the dynamic range of an IMS-MS instrument, particularly favoring targeted analysis in complex matrices. Alternatively, the analyte ions could be operated in a so-called trap-scan mode in which ions were trapped first in one of the channels and released sequentially for mobility analysis with an even higher resolution (ca. 210 for singly charged small organic molecules and ca. 590 for +15 myoglobin). Overall, this new UMA device would enable many new applications in omics studies with its high resolution and dynamic range, especially when using the filter-scan mode for scrutinizing analytes with very low concentrations under high chemical backgrounds.

Critical examination of gas-phase protein conformation/multimer ion formation by electrospray ion mobility-mass spectrometry.

The generally accepted view of protein structure in the gas-phase is that protein ions produced by electrospray ionization (ESI) exist in a number of different states, and the resulting charge state distribution (CSD) and ion mobility spectrum is interpreted as evidence for protein ions retaining some memory of solution-phase conformation. Even with the inclusion of ion mobility information, reports of protein ion structure in the gas-phase are oftentimes in disagreement not only within the discipline but also as interpreted by other gas-phase techniques. The focus of this work will be to correctly distinguish truly different ion conformations formed by ESI versus homomultimeric complexes with the same m/z. The concentration of cytochrome c in solution was varied over a wide range, and the multiply charged multimers (MCMs) present in the ion mobility/mass spectrum were unambiguously assigned by m/z selection and dissociation prior to ion mobility/mass spectrometry analysis. The results revealed false negatives for protein oligomer formation and false positives for protein conformational states and no evidence that gas-phase cytochrome c ions retain memory of solution-phase conformation, characteristics of great importance for structural biology. The results also suggest that the total IM-MS distribution for a protein is the complex result of individual MCMs either surviving until detection (undissociated) or dissociating into lower order multimers or a number of product ions for each m/z.

Kelvin spray ionization.

A novel self-powered dual spray ionization source has been developed for applications in mass spectrometry. This new source does not use any power supply and produces both positive and negative ions simultaneously. The idea behind this ionization source comes from the Kelvin water dropper. The source employs one or two syringes, two pneumatic sprays operated over a range of flow rates (0.15-15 µL min(-1)) and gas pressures (0-150 psi), and two double layered metal screens for ion formation. A variable electrostatic potential from 0 to 4 kV can be produced depending on solvent and gas flow rates that allow gentle ionization of compounds. There are several parameters that affect the performance during ionization of molecules including the flow rate of solvent, gas pressure, solvent acidity, position of spray and metal screens with respect to each other and distance between metal screens and the counter electrode. This ionization method has been successfully applied to solutions of peptides, proteins and non-covalent complexes. In comparison with ESI, the charge number of the most populated state is lower than that from ESI. It indicates that this is a softer ionization technique and it produces more protein ions with folded structures. The unique features of Kelvin spray ionization (KeSI) are that the method is self-powered and ionization occurs at very low potentials by providing very low internal energy to the ions. This advantage can be used for the ionization of very fragile molecules and investigation of non-covalent interactions.

Ion mobility-mass spectrometer interface for collisional activation of mobility separated ions.

An ion mobility-mass spectrometer (IM-MS) interface is described that can be employed to perform collisional activation and/or collision-induced dissociation (CID) with good transmission of mobility separated ions to the MS analyzer. The IM-MS interface consists of a stacked-ring ion guide design, where the field strength and pressure ratio can be operated such that structural rearrangement reactions and/or CID are achieved as a function of the effective ion temperature. The ion dynamics and collisional activation processes in the IM-MS interface are described as a function of the ion-neutral collisions, ion kinetic energies, and effective ion temperature. The applicability of the IM-CID-MS methodology to studies of peptide ion fragmentation is illustrated using a series of model peptides.

Gas-phase protein conformation/multimer ion formation by electrospray ion mobility-mass spectrometry: bovine insulin and ubiquitin.

Ion mobility-mass spectrometry (IMMS) is a very attractive method for studies in structural biology because of the ability of rapid isolation by nearly simultaneous m/z characterization and size separation, leading to an emergence of IMMS as a complimentary biochemical tool. Earlier, we developed a method based on varying the protein concentration in solution prior to electrospray ionization (ESI) with subsequent m/z selection and dissociation of protein multimers by IMMS of cytochrome c. The focus of this work will be to correctly distinguish truly different ion conformations formed by ESI versus homomultimeric complexes with the same m/z for well-studied proteins bovine ubiquitin and insulin. These proteins were chosen due to their large difference in solution phase structures: insulin tightly bound by disulfide linkages, and ubiquitin-a protein that may adopt a range of states from compact to extended. Our preliminary results, as with cytochrome c reveal false negatives for protein oligomer formation and false positives for protein conformational states. In addition, these results will be couched in terms of the need for quantification of IMMS analysis of proteins given the total area under IMMS peaks can also distinguish conformation versus aggregation as higher order oligomers have more mass per ion.This article is part of the themed issue 'Quantitative mass spectrometry'.

Analysis of Saccharides by the Addition of Amino Acids.

In this work, we present the detection sensitivity improvement of electrospray ionization (ESI) mass spectrometry of neutral saccharides in a positive ion mode by the addition of various amino acids. Saccharides of a broad molecular weight range were chosen as the model compounds in the present study. Saccharides provide strong noncovalent interactions with amino acids, and the complex formation enhances the signal intensity and simplifies the mass spectra of saccharides. Polysaccharides provide a polymer-like ESI spectrum with a basic subunit difference between multiply charged chains. The protonated spectra of saccharides are not well identified because of different charge state distributions produced by the same molecules. Depending on the solvent used and other ions or molecules present in the solution, noncovalent interactions with saccharides may occur. These interactions are affected by the addition of amino acids. Amino acids with polar side groups show a strong tendency to interact with saccharides. In particular, serine shows a high tendency to interact with saccharides and significantly improves the detection sensitivity of saccharide compounds. Graphical Abstract ᅟ.

The influence and utility of varying field strength for the separation of tryptic peptides by ion mobility-mass spectrometry.

The influence of field strength on the separation of tryptic peptides by drift tube-based ion mobility-mass spectrometry is reported. Operating the ion mobility drift tube at elevated field strengths (expressed in V cm(-1) torr(-1)) reduces separation times and increases ion transmission efficiencies. Several accounts in the literature suggest that performing ion mobility separation at elevated field strength can change the selectivity of ion separation. To evaluate the field strength dependant selectivity of ion mobility separation, we examined a data set of 65 singly charged tryptic peptide ion signals (mass range 500-2500 m/z) at six different field strengths and four different drift gas compositions (He, N2, Ar, and CH4). Our results clearly illustrate that changing the field strength from low field (15 V cm(-1) torr(-1)) to high field (66 V cm(-1) torr(-1)) does not significantly alter the selectivity or peak capacity of IM-MS. The implications of these results are discussed in the context of separation methodologies that rely on the field strength dependence of ion mobility for separation selectivity, e.g., high-field asymmetric ion mobility spectrometry (FAIMS).

The structure of gas-phase bradykinin fragment 1-5 (RPPGF) ions: an ion mobility spectrometry and H/D exchange ion-molecule reaction chemistry study.

Ion mobility-mass spectrometry (IM-MS) data is interpreted as evidence that gas-phase bradykinin fragment 1-5 (BK1-5, RPPGF) [M + H](+) ions exist as three distinct structural forms, and the relative abundances of the structural forms depend on the solvent used to prepare the matrix-assisted laser desorption ionization (MALDI) samples. Samples prepared from organic rich solvents (90% methanol/10% water) yield ions having an ion mobility arrival-time distribution (ATD) that is dominated by a single peak; conversely, samples prepared using mostly aqueous solvents (10% methanol/90% water) yield an ATD composed of three distinct peaks. The BK1-5 [M + H](+) ions were also studied by gas-phase hydrogen/deuterium (H/D) exchange ion-molecule reactions and this data supports our interpretation of the IM-MS data. Plausible structures for BK1-5 ions were generated by molecular dynamics (MD). Candidate MD-generated structures correlated to measured cross-sections suggest a compact conformer containing a beta-turn whereas a more extended, open form does not contain such an interaction. This study illustrates the importance of intra-molecular interactions in the stabilization of the gas-phase ions, and these results clearly illustrate that solution-phase parameters (i.e., MALDI sample preparation) greatly influence the structures of gas-phase ions.

Angiotensin II-acetylcholine noncovalent complexes analyzed with MALDI-ion mobility-TOF MS.

Matrix-assisted laser desorption ionization-ion mobility-orthogonal time-of-flight mass spectrometry (MALDI-IM oTOF MS) is a new technique that allows laser desorbed ion to be preseparated on the basis of their shape prior to mas analysis. Using this instrument, we tested the postulate that addition of a quaternary ammonium compound such as acetylcholine to the model phosphorylated peptide angio tensin II would enhance its detection by MALDI in two ways. First of all, the acetylcholine-peptide complex could ionize more efficiently than the bare phosphopeptide. Furthermore the ion mobility could separate the complex ion on the basis of its charge/volume from isobaric interferences, which would otherwise limit detection sensitivity.

Resolution equations for high-field ion mobility.

An extension of current mobility resolution equations as they apply to high-field ion mobility spectrometry is presented. The new resolution expression is applied to arrival time distributions for ions having a large range of ion mobilities and mass-to-charge ratios (m/z). The results indicate that the new equation can be utilized to predict the mobility resolution over a broader range of applied electric fields than previous ion mobility resolution expressions.

A study of peptide-peptide interactions using MALDI ion mobility o-TOF and ESI mass spectrometry.

Matrix-assisted laser desorption ionization ion mobility coupled to orthogonal time-of-flight mass spectrometry (MALDI-IM-oTOF MS) is evaluated as a tool for studying non-covalent complex (NCX) formation between peptides. The NCX formed between dynorphin 1-7 and Mini Gastrin I is used as a model system for comparison to previous MALDI experiments (Woods, A. S.; Huestis, M. A. J. Am. Soc. Mass Spectrom. 2001, 12, 88-96). The dynorphin 1-7/Mini Gastrin I complex is stable after more than a ms drift time through the He filled mobility cell. Furthermore, the effects of solution pH on NCX ion signal intensity is measured both by MALDI-IM-MS analysis and by nanoelectrospray mass spectrometry. When compared to the previous MALDI study this work shows that all three techniques give similar results. In addition, fragmentation can be observed from of the non-covalent complex parent ion that occurs prior to TOF mass analysis but after mobility separation, thus providing NCX composition information.

Peak capacity of ion mobility mass spectrometry: the utility of varying drift gas polarizability for the separation of tryptic peptides.

Ion mobility mass spectrometry (IM-MS) peptide mass mapping experiments were performed using a variety of drift gases (He, N2, Ar and CH4). The drift gases studied cover a range of polarizabilities ((0.2-2.6) x 10(-24) cm3) and the peak capacities obtained for tryptic peptides in each gas are compared. Although the different gases exhibit similar peak capacities (5430 (Ar) to 7580 (N2)) in some cases separation selectivity presumably based on peptide conformers (or conformer populations), is observed. For example the drift time profiles observed for some tryptic peptide ions from aldolase (rabbit muscle) show a dependence on drift gas. The transmission of high-mass ions (m/z > 2000) is also influenced by increased scattering cross-section of the more massive drift gases. Consequently the practical peak capacity for IM-MS separation cannot be assumed to be solely a function of resolution and the ability of a gas to distribute signals in two-dimensional space; rather, peak capacity estimates must account for the transmission losses experienced for peptide ions as the drift gas mass increases.

Peak capacity of ion mobility mass spectrometry: separation of peptides in helium buffer gas.

Advances in the field of proteomics depend upon the development of high-throughput separation methods. Ion mobility-mass spectrometry is a fast separation method (separations on the millisecond time-scale), which has potential for peptide complex mixture analysis. Possible disadvantages of this technique center around the lack of orthogonality between separation based on ion mobility and separation based on mass. In order to examine the utility of ion mobility-mass spectrometry, the peak capacity (phi) of the technique was estimated by subjecting a large dataset of peptides to linear regression analysis to determine an average trend for tryptic peptides. This trend-line, along with the deviation from a linear relationship observed for this dataset, was used to define the separation space for ion mobility-mass spectrometry. Using the maximum deviation found in the dataset (+/-11%) the peak capacity of ion mobility-mass spectrometry is approximately 2600 peptides. These results are discussed in light of other factors that may increase the peak capacity of ion mobility-mass spectrometry (i.e. multiple trends in the data resulting from multiple classes of compounds present in a sample) and current liquid chromatography approaches to complex peptide mixture analysis.

Increasing the Performance of Portable Ion Mobility Analyzers: Development of the Periodic Focusing Differential Mobility Analyzer (PFDMA).

Ion mobility spectrometry (IMS) as a stand-alone technique has become increasingly important for applications in security, defense, and environmental monitoring, and also in biological applications such as molecular structure and -omic analysis when combined with mass spectrometry. Yet, the majority of these devices are drift cell based and limited by low duty cycles because of ion gating. Differential Mobility Analyzers (DMAs) are attractive alternatives due to their continuous ion transmission and success in analyzing aerosol particles in real time environmental tests. But, the resolution of a DMA is low due to difficulties in achieving laminar gas flow, low sample gas flow to sheath gas flow ratio, and high velocity sheath gas using small pumps, if portability is a concern. To overcome these challenges, we will introduce a new ion mobility spectrometer that increases the amount of work done on the ions during separation by introducing an electric field opposing the gas flow direction while simultaneously preserving laminar gas flow. The development of the Periodic Focusing Differential Mobility Analyzer (PFDMA) can lead to a portable device that exhibits both high resolution and sensitivity, to meet the needs of today's expanding applications.

Triboelectric spray ionization.

Triboelectric spray ionization (TESI) is a variation of electrospray ionization (ESI) using common instrumental components, including gas flow, solvent flow rate and heat, the only difference being the use of a high-voltage power supply for ESI or a static charge for TESI. The ionization of solvent or analyte is due to the electrostatic potential difference formed between the spray electrode and counter electrode. The ion source contains a pneumatic spray operated over a range of flow rates (0.15-1.5 µl/min) and gas pressures (0-100). This new design contains a standalone spray assembly and an optional metal mesh in front of the spray. There are several parameters that affect the performance during ionization of molecules including the flow rate of solvent, gas pressure, temperature, solvent acidity, distance and potential difference between emitter and counter electrode. A variable electrostatic potential can be applied for higher ionization efficiency. The new ionization method was successfully applied to solutions of various proteins under different conditions. The same charge-state distributions compared to other ESI techniques are observed for all the protein samples. The unique feature of TESI is very efficient spraying by using a natural electrostatic potential even at the potential that a human body can produce. This provides very gentle ionization efficiency of peptides and proteins in different solvents.

Kinetics of surface-induced dissociation of N(CH3)4(+) and N(CD3)4(+) using silicon nanoparticle assisted laser desorption/ionization and laser desorption/ionization.

The implementation of surface-induced dissociation (SID) to study the fast dissociation kinetics (sub-microsecond dissociation) of peptides in a MALDI TOF instrument has been reported previously. Silicon nanoparticle assisted laser desorption/ionization (SPALDI) now allows the study of small molecule dissociation kinetics for ions formed with low initial source internal energy and without MALDI matrix interference. The dissociation kinetics of N(CH(3))(4)(+) and N(CD(3))(4)(+) were chosen for investigation because the dissociation mechanisms of N(CH(3))(4)(+) have been studied extensively, providing well-characterized systems to investigate by collision with a surface. With changes in laboratory collision energy, changes in fragmentation timescale and dominant fragment ions were observed, verifying that these ions dissociate via unimolecular decay. At lower collision energies, methyl radical (CH(3)) loss with a sub-microsecond dissociation rate is dominant, but consecutive H loss after CH(3) loss becomes dominant at higher collision energies. These observations are consistent with the known dissociation pathways. The dissociation rate of CH(3) loss from N(CH(3))(4)(+) formed by SPALDI and dissociated by an SID lab collision energy of 15 eV corresponds to log k = 8.1, a value achieved by laser desorption ionization (LDI) and SID at 5 eV. The results obtained with SPALDI SID and LDI SID confirm that (1) the dissociation follows unimolecular decay as predicted by RRKM calculations; (2) the SPALDI process deposits less initial energy than LDI, which has advantages for kinetics studies; and (3) fluorinated self-assembled monolayers convert about 18% of laboratory collision energy into internal energy. SID TOF experiments combined with SPALDI and peak shape analysis enable the measurement of dissociation rates for fast dissociation of small molecules.

A novel approach to collision-induced dissociation (CID) for ion mobility-mass spectrometry experiments.

Collision induced dissociation (CID) combined with matrix assisted laser desorption ionization-ion mobility-mass spectrometry (MALDI-IM-MS) is described. In this approach, peptide ions are separated on the basis of mobility in a 15 cm drift cell. Following mobility separation, the ions exit the drift cell and enter a 5 cm vacuum interface with a high field region (up to 1000 V/cm) to undergo collisional activation. Ion transmission and ion kinetic energies in the interface are theoretically evaluated accounting for the pressure gradient, interface dimensions, and electric fields. Using this CID technique, we have successfully fragmented and sequenced a number of model peptide ions as well as peptide ions obtained by a tryptic digest. This instrument configuration allows for the simultaneous determination of peptide mass, peptide-ion sequence, and collision-cross section of MALDI-generated ions, providing information critical to the identification of unknown components in complex proteomic samples.

Observation of conserved solution-phase secondary structure in gas-phase tryptic peptides.

Results from ion mobility studies of tryptic peptides suggest that, in some cases, the gas-phase structures can be related to the solution-phase structure of the parent protein. The interpretation of ion mobility measurements is supported by results from molecular modeling and H/D exchange experiments on the same peptides. This study clearly illustrates the utility of IM-MS for screening complex mixtures for peptides having intrinsically stable secondary/tertiary structures, and/or posttranslational modification.

A fundamental introduction to ion mobility mass spectrometry applied to the analysis of biomolecules.

Chemists are constantly striving for techniques that add dimensions of orthogonality with increased throughput and sample complexity. Ion mobility spectrometry (IM) is a gas-phase separation method that adds new dimensions to mass spectrometry (MS). IM separates gas-phase ions based on their collision cross-section and can be coupled with time-of-flight (TOF) mass spectrometry to yield a powerful tool used in the identification and characterization of proteins and peptides. A fundamental introduction to IM is presented with a focus on resolution, sensitivity, and orthogonality coupled with TOF-MS.

Distinguishing between phosphorylated and nonphosphorylated peptides with ion mobility-mass spectrometry.

Mass spectrometry has become an indispensable tool in identifying post-translationally modified proteins, but multiple peptide mass-mapping/peptide-sequencing experiments are required to answer questions involving the site and type of modification present. Here, we apply ion mobility-mass spectrometry (IM-MS), a high-throughput analysis method having high selectivity and sensitivity, to the challenge of identifying phosphorylated peptides. Ion mobility separation is based on the collision cross-section of the ion. Phosphorylation can result in a conformational change in gas-phase peptide ions, which can be detected by IM. To demonstrate this point, a peptide mixture containing a variety of peptide sequences is examined with IM-MS and molecular dynamics calculations. During the course of these studies, two classes of phosphopeptide were identified: (i) phosphorylated peptide ions that have conformers that differ from the nonphosphorylated ion and (ii) phosphorylated peptide ions that have conformations that are very similar to the nonphosphorylated peptide. The utility of IM-MS peptide mass mapping for identifying both types of phosphorylated peptides is discussed.