Rapid separation and quantitative analysis of peptides using a new nanoelectrospray- differential mobility spectrometer-mass spectrometer system

Differential mobility spectrometry (DMS) (see Buryakov, I. A.; Krylov, E. V.; Nazarov, E. G.; Rasulev, U. Kh. Int. J. Mass Spectrom. Ion Processes 1993, 128, 143-148), also commonly referred to as high-field asymmetric waveform ion mobility spectrometry (FAIMS) (see Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszcyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094-4105), is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal-to-noise, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and the potential for rapid analyte quantitation. In this report, we investigate the use of our nanoESI-DMS-MS system to demonstrate differential mobility separation of peptides. The formation of higher order peptide aggregate ions (ion complexes) via electrospray ionization and the negative impact this has on DMS peptide separation are examined. The successful use of differential mobility drift gas modifiers (dopants) to reduce aggregate ion size and improve DMS peptide ion separation is presented. Following optimization of DMS peptide separation conditions, we examined next the feasibility of a new analytical platform which uses direct sample infusion with nanoESI-DMS-MS for ultrarapid analyte quantitation. Quantitation of a selected peptide from a semicomplex peptide mixture is presented. Initial feasibility results with this new approach demonstrate good accuracy and reproducibility, as well as an absolute mass sensitivity of 6.8 amol and a minimum dynamic range of 2500 for the peptide of interest. This report offers a first look at utilizing nanoESI-DMS-MS to create an ultrarapid (under 5 s) quantitative analysis platform and its potential in the high-throughput arena. Each ion separation technique, DMS and MS, offers orthogonal ion separation to one another, enhancing the overall specificity for this quantitative approach.     

Ion trap/ion mobility/quadrupole/time-of-flight mass spectrometry for peptide mixture analysis

An ion trap/ion mobility/quadrupole/time-of-flight mass spectrometer has been developed for the analysis of peptide mixtures. In this approach, a mixture of peptides is electrosprayed into the gas phase. The mixture of ions that is created is accumulated in an ion trap and periodically injected into a drift tube where ions separate according to differences in gas-phase ion mobilities. Upon exiting the drift tube, ions enter a quadrupole mass filter where a specific mass-to-charge (m/z) ratio can be selected prior to collisional activation in an octopole collision cell. Parent and fragment ions that exit the collision cell are analyzed using a reflectron geometry time-of-flight mass spectrometer. The overall configuration allows different species to be selected according to their mobilities and m/z ratios prior to collision-induced dissociation and final MS analysis. A key parameter in these studies is the pressure of the target gas in the collision cell. Above a critical pressure, the well-defined mobility separation degrades. The approach is demonstrated by examining a mixture of tryptic digest peptides of ubiquitin.     

A tandem ion trap/ion mobility spectrometer

A tandem quadrupole ion trap/ion mobility spectrometer (QIT/IMS) has been constructed for structural analysis based on the gas-phase mobilities of mass-selected ions. The instrument combines the ion accumulation, manipulation, and mass-selection capabilities of a modified ion trap mass spectrometer with gas-phase electrophoretic separation in a custom-built ion mobility drift cell. The quadrupole ion trap may be operated as a conventional mass spectrometer, with ion detection using an off-axis dynode/multiplier arrangement, or as an ion source for the IMS drift cell. In the latter case, pulses of ions are ejected from the trap and transferred to the drift cell where mobility in the presence of helium buffer gas is determined by the collision cross section of the ion. Ions traversing the drift cell are detected by an in-line electron multiplier and the data processed with a multichannel scaler. Preliminary data are presented on instrumental performance characteristics and the application of QIT/ IMS to structural and conformational studies of aromatic ions and protonated amine/crown ether noncovalent complexes generated via ion/molecule reactions in the ion trap.     

Separation of isomeric peptides using electrospray ionization/high-resolution ion mobility spectrometry

In this paper, the first examples of baseline separation of isomeric macromolecules by electrospray ionization/ion mobility spectrometry (ESI/IMS) at atmospheric pressure are presented. The behavior of a number of different isomeric peptides in the IMS was investigated using nitrogen as a drift gas. The IMS was coupled to a quadrupole mass spectrometer, which was used for identification and selective detection of the electrosprayed ions. The mobility data were used to determine their average collision cross sections. The gas-phase ions of isomeric peptides were found to have different collision cross sections. In all cases, doubly charged ions exhibited significantly (8-20%) larger collision cross sections than the respective singly charged species. The analysis of mixtures of the isomeric peptides clearly demonstrated the capability of IMS to separate gas-phase peptide ions due to small differences in their conformational structures, which cannot be determined by mass spectrometry. An actual resolving power of 80 was achieved for two doubly charged reversed sequenced pentapeptides. Baseline separation was provided for ions differing by only 2.5% in their measured collision cross sections; partial separation was shown for isomeric ions exhibiting differences as small as 1.1%.     

Gas-phase chiral separations by ion mobility spectrometry

This article introduces the concept of chiral ion mobility spectrometry (CIMS) and presents examples demonstrating the gas-phase separation of enantiomers of a wide range of racemates including pharmaceuticals, amino acids, and carbohydrates. CIMS is similar to traditional ion mobility spectrometry, where gas-phase ions, when subjected to a potential gradient, are separated at atmospheric pressure due to differences in their shapes and sizes. In addition to size and shape, CIMS separates ions based on their stereospecific interaction with a chiral gas. In order to achieve chiral discrimination by CIMS, an asymmetric environment was provided by doping the drift gas with a volatile chiral reagent. In this study (S)-(+)-2-butanol was used as a chiral modifier to demonstrate enantiomeric separations of atenolol, serine, methionine, threonine, methyl alpha-glucopyranoside, glucose, penicillamine, valinol, phenylalanine, and tryptophan from their respective racemic mixtures.     

Producing highly charged ions without solvent using laserspray ionization: a total solvent-free analysis approach at atmospheric pressure

First examples of highly charged ions in mass spectrometry (MS) produced from the solid state without using solvent during either sample preparation or mass measurement are reported. Matrix material, matrix/analyte homogenization time and frequency, atmospheric pressure (AP) to vacuum inlet temperature, and mass analyzer ion trap conditions are factors that influence the abundance of the highly charged ions created by laserspray ionization (LSI). LSI, like matrix-assisted laser desorption/ionization (MALDI), uses laser ablation of a matrix/analyte mixture from a surface to produce ions. Preparing the matrix/analyte sample without the use of solvent provides the ability to perform total solvent-free analysis (TSA) consisting of solvent-free ionization and solvent-free gas-phase separation using ion mobility spectrometry (IMS) MS. Peptides and small proteins such as non-β-amyloid components of Alzheimer's disease and bovine insulin are examples in which LSI and TSA were combined to produce multiply charged ions, similar to electrospray ionization, but without the use of solvent. Advantages using solvent-free LSI and IMS-MS include simplicity, rapid data acquisition, reduction of sample complexity, and the potential for an enhanced effective dynamic range. This is achieved by more inclusive ionization and improved separation of mixture components as a result of multiple charging.     

Separation of ions from explosives in differential mobility spectrometry by vapor-modified drift gas

Differential mobility spectrometry (DMS) of nitro-organic explosives and related compounds exhibited the expected product ions of M- or M x NO2- from atmospheric pressure chemical ionization reactions in purified air at 100 degrees C. Peaks in the differential mobility spectra for these ions were confined to a narrow range of compensation voltages between -1 to +3 V which arose through a low dependence of mobility for the ions in electric fields at E/N values between 0 and 120 Td (1 Td = 10(-17) V cm2). The field dependence of ions, described as an alpha parameter, ranged from -0.005 to 0.02 at a separation field of 100 Td. The alpha parameter could be controlled through the addition of organic vapors into the drift gas and was increased to 0.08-0.24 with 1000 ppm of methylene chloride in the drift gas. This modification of the drift gas resulted in compensation voltages of +3 to +21 V for peaks. The improved separation of peaks was consistent with a model of ion characterization by DeltaK or Kl - Kh, where Kl is the mobility coefficient of ions clustered with vapor neutrals during the low-field portion of the separation field waveform and Kh is for the same core ion when heated and declustered during the high-field portion of waveform.     

Gas-phase proton-transfer chemistry coupled with TOF mass spectrometry and ion mobility-MS for the facile analysis of poly(ethylene glycols) and PEGylated polypeptide conjugates

Gas-phase ion/molecule chemistry has been combined with ion mobility separation and time-of-flight mass spectrometry to enable the characterization of large poly(ethylene glycol)s (PEGs) and PEGylated molecules (>40 kDa). A facile method is presented in which gas-phase superbases are reacted in the high-pressure source region of commercial TOF mass spectrometers to manipulate the charge states of large ions generated by electrospray ionization (ESI). Charge stripping decreases the spectral congestion typically observed in ESI mass spectra of high molecular weight polydisperse PEGylated molecules. From these data, accurate average molecular weights and molecular weight distributions for synthetic polymers and PEGylated proteins are determined. The average MW measured for PEGylated Granulocyte colony-stimulating factor (rh-GCSF, 40 726.2 Da) is in good agreement with the theoretical value, and a 16 Da mass shift is easily observed in the spectrum of an oxidized form of the heterogeneous PEGylated protein. Ion mobility separations can fractionate PEGs of different chain length; when coupled with charge stripping ion/molecule reactions, ion mobility mass spectrometry (IMMS) offers several analytical advantages over mass spectrometry alone for the characterization of large PEGylated molecules including enhanced dynamic range, increased sensitivity, and specificity. Low abundance free PEG in a PEGylated peptide preparation, which is not directly detectable by mass spectrometry, can be easily observed and accurately quantified with gas-phase ion/molecule chemistry combined with ion mobility mass spectrometry.     

Using different drift gases to change separation factors (alpha) in ion mobility spectrometry

The use of different drift gases to alter separation factors (alpha) in ion mobility spectrometry has been demonstrated. The mobility of a series of low molecular weight compounds and three small peptides was determined in four different drift gases. The drift gases chosen were helium, argon, nitrogen, and carbon dioxide. These drift gases provide a range of polarizabilities and molecular weights. In all instances, the compounds showed the greatest mobility in helium and the lowest mobility in carbon dioxide; however the percentage change of mobility for each compound was different, effectively changing the alpha value. The alpha value changes were primarily due to differences in drift gas polarizability but were also influenced by the mass of the drift gas. In addition, gas-phase ion radii were calculated in each of the different drift gases. These radii were then plotted against drift gas polarizability producing linear plots with r2 values greater than 0.99. The intercept of these plots provides the gas-phase radius of an ion in a nonpolarizing environment, whereas the slope is indicative of the magnitude of the ion's mobility change related to polarizability. It therefore, should be possible to separate any two compounds that have different slopes with the appropriate drift gas.     

Voltage sweep ion mobility spectrometry

Ion mobility spectrometry (IMS) is a rapid, gas-phase separation technique that exhibits excellent separation of ions as a standalone instrument. However, IMS cannot achieve optimal separation power with both small and large ions simultaneously. Similar to the general elution problem in chromatography, fast ions are well resolved using a low electric field (50-150 V/cm), whereas slow drifting molecules are best separated using a higher electric field (250-500 V/cm). While using a low electric field, IMS systems tend to suffer from low ion transmission and low signal-to-noise ratios. Through the use a novel voltage algorithm, some of these effects can be alleviated. The electric field was swept from low to high while monitoring a specific drift time, and the resulting data were processed to create a 'voltage-sweep' spectrum. If an optimal drift time is calculated for each voltage and scanned simultaneously, a spectrum may be obtained with optimal separation throughout the mobility range. This increased the resolving power up to the theoretical maximum for every peak in the spectrum and extended the peak capacity of the IMS system, while maintaining accurate drift time measurements. These advantages may be extended to any IMS, requiring only a change in software.     

Ultrasensitive identification of localization variants of modified peptides using ion mobility spectrometry

Localization of the modification sites on peptides is challenging, particularly when multiple modifications or mixtures of localization isomers (variants) are involved. Such variants commonly coelute in liquid chromatography and may be undistinguishable in tandem mass spectrometry (MS/MS) for lack of unique fragments. Here, we have resolved the variants of singly and doubly phosphorylated peptides employing drift tube ion mobility spectrometry (IMS) coupled to time-of-flight mass spectrometry. Even with a moderate IMS resolving power of ～80-100, substantial separation was achieved for both 2+ and 3+ ions normally generated by electrospray ionization, including for the variants indistinguishable by MS/MS. Variants often exhibit a distribution of 3-D conformers, which can be adjusted for optimum IMS separation by prior field heating of ions in a funnel trap. The peak assignments were confirmed using MS/MS after IMS separation, but known species could be identified using just the ion mobility "tag". Avoiding the MS/MS step lowers the detection limit of localization variants to <100 amol, an order of magnitude better than that provided by electron transfer dissociation in an Orbitrap MS.     

Mobility labeling for parallel CID of ion mixtures

An ion mobility/mass spectrometry technique has been developed to record collision-induced dissociation patterns for multiple ions in a parallel fashion. In this approach, a mixture of ions is separated in a drift tube on the basis of differences in mobilities through a buffer gas. As the ions exit the drift tube, they are accelerated into a collision cell and the ensuing fragment ions are dispersed by differences in mass-to-charge (m/z) ratios in a time-of-flight mass spectrometer. Fragment ions that are formed in the collision cell have drift times that are coincident with their antecedent parent ions, allowing the origin of all fragments formed from the mixture of ions to be determined. The approach is demonstrated by examining fragmentation patterns of the [M + H]+ parent and a series of a-, b-, and y-type fragments of [D-Ala2,3]methionine enkephalin.     

Shift reagents for multidimensional ion mobility spectrometry-mass spectrometry analysis of complex peptide mixtures: evaluation of 18-crown-6 ether complexes

18-Crown-6 ether (18C6) is evaluated as a shift reagent for multidimensional ion mobility spectrometry-mass spectrometry (IMS-IMS-MS) analyses of tryptic protein digests. In this approach, 18C6 is spiked into the solution-phase mixture and noncovalent peptide-crown ion complexes are formed by electrospraying the mixture into the gas phase. After an initial mobility separation in the first IMS drift region, complexes of similar mobility are selected and dissociated via collisional activation prior to entering the second drift region. These dissociation products (including smaller complexes, naked peptide ions, charge transfer products, and fragment ions) differ in mobility from their precursor ion complexes and (in favorable cases) from one another, allowing the mixture to resolve further in the second IMS region. We estimate an IMS-IMS peak capacity of ~2400 when shift reagents are employed. The approach is illustrated by examining a tryptic digest of cytochrome c and by identifying a peptide out of a complex mixture obtained by digestion of human plasma proteins. Disadvantages arising from increased complexity of data sets as well as other advantages of this approach are considered.     

Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry

One of the major factors governing the "top-down" sequence analysis of intact multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions. To more fully understand this effect, electrospray ionization coupled to a quadrupole ion trap mass spectrometer, collision-induced dissociation, and gas-phase ion/ion reactions have been employed to examine the fragmentation of the [M + 12H]12+ to [M + H]+ ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed cleavage at aspartic acid residues was observed. At intermediate charge states, (+7, +8, and +9), extensive nonspecific fragmentation of the protein backbone was observed, with 50% sequence coverage obtained from the [M + 8H]8+ ion alone. At high charge states, (+10, +11, +12), the single dominant channel that was observed was the preferential fragmentation of a single proline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton model", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquitin in the gas phase, examined by ion mobility and hydrogen/deuterium exchange measurements.     

Surface-induced dissociation of ion mobility-separated noncovalent complexes in a quadrupole/time-of-flight mass spectrometer

A custom in-line surface-induced dissociation (SID) device has been incorporated into a commercial ion mobility quadrupole/time-of-flight mass spectrometer in order to provide an alternative and potentially more informative activation method than the commonly used collision-induced dissociation (CID). Complicated sample mixtures can be fractionated by ion mobility (IM) and then dissociated by CID or SID for further structural analysis. Interpretation of SID spectra for cesium iodide clusters was greatly simplified with IM prior to dissociation because products originating from different precursors and overlapping in m/z but separated in drift time can be examined individually. Multiple conformations of two protein complexes, source-activated transthyretin tetramer and nativelike serum amyloid P decamer, were separated in ion mobility and subjected to CID and SID. CID spectra of the mobility separated conformations are similar. However, drastic differences can be observed for SID spectra of different conformations, implying different structures in the gas phase. This work highlights the potential of utilizing IM-SID to study quaternary structures of protein complexes and provides information that is complementary to our recently reported SID-IM approach.     

Electrospray ionization high-resolution ion mobility spectrometry for the detection of organic compounds, 1. Amino acids

Our aim in this investigation was to demonstrate the potential of the high-resolution electrospray ionization ion mobility spectrometry (ESI-IMS) technique as an analytical separation tool in analyzing biomolecular mixtures to pursue astrobiological objectives of searching for the chemical signatures of life during an in-situ exploration of solar system bodies. Because amino acids represent the basic building blocks of life, we used common amino acids to conduct the first part of our investigation, which is being reported here, to demonstrate the feasibility of using the ESI-IMS technique for detection of the chemical signatures of life. The ion mobilities of common amino acids were determined by electrospray ionization ion mobility spectrometry using three different drift gases (N2, Ar, and CO2). We demonstrated that the selectivity can be vastly improved in ion mobility spectroscopy (IMS) in detecting organic molecules by using different drift gases. When a judicial choice of drift gas is made, a vastly improved separation of two different amino acid ions resulted. It was found that each of the studied amino acids could be uniquely identified from the others, with the exception of alanine and glycine, which were never separable by more then 0.1 ms. This unique identification is a result of the different polarizabilities of the various drift gases. In addition, a better separation was achieved by changing the drift voltage in successive experimental runs without significantly degrading the resolution. We also report the result of our analysis of liquid samples containing mixtures of amino acids.     

Probing electrospray ionization dynamics using differential mobility spectrometry: the curious case of 4-aminobenzoic Acid

Here, we present the separation of two ions that differ only by the site of protonation of the analyte molecule using differential mobility spectrometry (DMS). Protonated 4-aminobenzoic acid molecules (4-ABA) generated by positive-mode electrospray ionization [ESI(+)] can exist with the proton residing on either the amine nitrogen (N-protonated) or the carboxylic acid oxygen (O-protonated), and the protonation site can differ on the basis of the solvent system used. In this study, we demonstrate the identification and separation of N- and O-protonated 4-ABA using DMS, with structural assignments verified by: (1) the presence of distinct peaks in the DMS ionogram, (2) the observed effects resulting from altering the ESI(+) solvent system, (3) the observed (13)C NMR chemical shifts arising from altering the solvent system, (4) the observation of distinct MS/MS fragmentation patterns for the two DMS-separated ions, (5) the unique hydrogen-deuterium exchange behavior for these ions, and (6) the fundamental behavior of these two ions within the DMS cell, linked back to the structural differences between the two protonated forms.     

Enhanced mixture analysis of poly(ethylene glycol) using high-field asymmetric waveform ion mobility spectrometry combined with fourier transform ion cyclotron resonance mass spectrometry

The combination of high-field asymmetric waveform ion mobility spectrometry (FAIMS) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) makes possible lower detection limits, increased sensitivity, and increased dynamic range in the analysis of poly(ethylene glycol) (PEG) samples of low molecular weight. The signal gain obtained using FAIMS depends on ion identity, with a range between 1.8x and 14x obtained for various molecular ions of PEG 600. A 1.7-fold reduction in noise is obtained using FAIMS due to the elimination of chemical noise. The improved detection performance is predominantly due to a reduction in adverse Coulomb effects as a result of ions being selectively introduced into the mass spectrometer. The high ion transmission obtained using FAIMS combined with the high sensitivity of FTICR-MS detection make possible separation of multiple gas-phase conformers of PEG molecular cations that have low abundance (less than 0.2% relative abundance) and that have not been detected previously. Mixed dications of PEG that have the same nominal mass but differ by the number polymer subunits (m/Delta m up to 25,000) can be separately introduced into the mass spectrometer using FAIMS. Interactions of the carrier gas with the metal ions that are attached to the PEG molecules appear to be the most significant factor in these FAIMS separations.     

Mass analysis of mobility-selected ion populations using dual gate, ion mobility, quadrupole ion trap mass spectrometry

An electrospray ionization, dual gate, ion mobility, quadrupole ion trap mass spectrometer (ESI-DG-IM-QIT-MS) was constructed and evaluated for its ability to select mobility-filtered ions prior to mass analysis. While modification of the common signal-averaged ion mobility experiment was required, no modifications to the QIT were necessary. The dual gate scanning mode of operation was used to acquire mobility spectra, whereas the single mobility monitoring experiment selectively filtered ions for concentration and subsequent fragmentation within the QIT. Ion mobility separation of positively charged peptides and negatively charged carbohydrates, followed by MS fragmentation, was demonstrated. For a 1-min acquisition time, it was possible to obtain complete de novo sequence information for the examined peptides. Fragmentation of the negative carbohydrate chlorine adducts yielded ions characteristic of cross-ring and glycosidic bond cleavage. Previous unions of atmospheric pressure ion mobility and mass spectrometry have been limited in their ability to reproducibly obtain MSn data for mobility separation ions. The union of high-pressure ion mobility with quadrupole ion trap mass spectrometry presents the unique opportunity to obtain more detailed information regarding the chemistries of gas-phase ions.