Simple coupling of gas chromatography to electrospray ionization mass spectrometry

A simple method for direct coupling of gas chromatography (GC) with electrospray ionization mass spectrometry (ESI/MS) has been developed. The outlet of the GC capillary column was placed between the ESI needle and the atmospheric pressure ionization (API) source of a mass spectrometer. The ionization occurs via dissolution of neutral compounds into the charged ESI droplet followed by ion evaporation or via a gas-phase proton transfer reaction between a protonated solvent molecule and an analyte. The mass spectra of organic volatile compounds showed abundant protonated molecules with little fragmentation, being very similar to those produced by normal liquid ESI. The quantitative performance of the system was evaluated by determining the limit of detection (LOD), linearity ( r (2)), and repeatability (RSD). The GC-ESI/MS method was shown to be stable, providing high sensitivity and good quantitative performance.     

Secondary electrospray ionization ion mobility spectrometry/mass spectrometry of illicit drugs

A secondary electrospray ionization (SESI) method was developed as a nonradioactive ionization source for ion mobility spectrometry (IMS). This SESI method relied on the gas-phase interaction between charged particles created by electrospray ionization (ESI) and neutral gaseous sample molecules. Mass spectrometry (MS) was used as the detection method after ion mobility separation for ion identification. Preliminary investigations focussed on understanding the ionization process of SESI. The performance of ESI-IMS and SESI-IMS for illicit drug detection was evaluated by determining the analytical figures of merit. In general, SESI had a higher ionization efficiency for small volatile molecules compared with the electrospray method. The potential of developing a universal interface for both GC- and LC-MS with an addition stage of mobility separation was demonstrated.     

Zeptomole-sensitivity electrospray ionization--Fourier transform ion cyclotron resonance mass spectrometry of proteins

Methods are being developed for ultrasensitive protein characterization based upon electrospray ionization (ESI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The sensitivity of a FTICR mass spectrometer equipped with an ESI source depends on the overall ion transmission, which combines the probability of ionization, transmission efficiency, and ion trapping in the FTICR cell. Our developments implemented in a 3.5 tesla FTICR mass spectrometer include introduction and optimization of a newly designed electrodynamic ion funnel in the ESI interface, improving the ion beam characteristics in a quadrupole-electrostatic ion guide interface, and modification of the electrostatic ion guide. These developments provide a detection limit of approximately 30 zmol (approximately 18,000 molecules) for proteins with molecular weights ranging from 8 to 20 kDa.     

Molecular weight distributions of asphaltenes and deasphaltened oils studied by laser desorption ionization and ion mobility mass spectrometry

Laser desorption ionization (LDI) and ion mobility mass spectrometry (IM-MS) are applied to study molecular weight distribution and cross sections of petroleum asphaltene (ASPH) and deasphaltened oils (DAO). Ion mobility data confirmed the presence of gas-phase aggregation in LDI experiments. Most of the molecules with MW > 3000 g/mol in LDI result from gas-phase aggregation. Two-dimensional (2D) IM-MS trend lines are compared with model polymer systems to confirm the order of cross sections (polywax > polystyrene > DAO > ASPH > fullerenes), and these data illustrate that ASPH has a more condensed average structure than DAO.     

Screening and identification of acidic carbohydrates in bovine colostrum by using ion/molecule reactions and Fourier transform ion cyclotron resonance mass spectrometry: specificity toward phosphorylated complexes

A screening method was developed for the identification of acidic saccharides from biological mixtures utilizing gas-phase derivatization and mass spectrometry. Phosphorylated compounds were differentiated from other acidic species by exploiting the selective reactivity of chlorotrimethylsilane with the phosphate ions (phosphorylated compounds shift by 72 Da, allowing rapid compound detection). A 13-component mock mixture was used to demonstrate the viability of the method, and a detection limit of 600 nM (30 fmol) was determined. This method was applied to the identification of acidic compounds from bovine colostrum. To further verify the selectivity of the ion/molecule reaction, exact mass measurements were used to determine the elemental composition of 14 compounds. Eight novel acidic carbohydrate species were observed in bovine colostrum, six of which have never been reported previously in milks. Tandem mass spectrometric experiments allowed compound characterization for two of these components.     

Characterization of gas-phase molecular interactions on differential mobility ion behavior utilizing an electrospray ionization-differential mobility-mass spectrometer system

Differential mobility spectrometry (DMS) 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/noise ratios, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and potential for rapid analyte quantitation. The introduction of a new ESI-DMS-MS system and its utilization to aid in the understanding of DMS separation theory is described. A current contribution to DMS separation theory is one of an association/dissociation process between ions/molecules in the gas phase during the differential mobility separation. A model study was designed to investigate the molecular dynamics and chemical factors influencing the theorized association/dissociation process, and the mechanisms by which these gas-phase interactions affect an ion's DM behavior. Five piperidine analogues were selected as model analytes, and three alcohol drift gas dopants/modifiers were used to interrogate the analyte ions in the gas phase. Two proposed DMS separation mechanisms, introduced as Core and Fa?ade, corresponding to strong and weak attractions between ions/molecules in the gas phase, are detailed. The proposed mechanisms provide explanation for the observed changes in analyte separation by the various drift gas modifiers. Molecular modeling of the proposed mechanisms provides supportive data and demonstrates the potential for predictive optimization of analyte separation based on drift gas modifier effects.     

FTMS structure elucidation of natural products: application to muraymycin antibiotics using ESI multi-CHEF SORI-CID FTMS(n), the top-down/bottom-up approach,and HPLC ESI capillary-skimmer CID FTMS

The molecular formulas for the structures and substructures of muraymycin antibiotics A1 (C52H90N14O19, MW 1214) and B1 (C49H83N11O18, MW 1113) were determined using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The muraymycin A1 and B1 structures were elucidated by utilizing capillary-skimmer fragmentation with up to five stages of mass spectrometry (MS5). Multi-CHEF, a multiple ion isolation method, was used at each stage of MS(n) to isolate a parent ion and up to four reference ions, for exact-mass calibration. The parent ions were fragmented by SORI-CID and the product ions internally calibrated with average absolute mass errors less than 1 ppm at each stage in the fragmentation processes. Using the top-down/bottom-up approach, the molecular formulas for the antibiotics were determined by summing the elemental formulas of the neutral losses, obtained by measuring the mass differences (<500 Da) between the genetically related sequential parent ion masses in the MS(n) spectra, with the unique elemental formula of the lowest parent ion mass (<500 Da). The structures of 12 additional compounds in the muraymycin complex were elucidated using HPLC ESI capillary-skimmer CID FTMS by correlating their fragmentation patterns with those of muraymycins A1 and B1. Sequential neutral losses of an aminosugar, a valine, a uridine, and an ester fatty acid from the muraymycin parent ions provided diagnostic fragments for characterization.     

Gas-phase electrophoretic molecular mobility analysis of size and stoichiometry of complexes of a common cold virus with antibody and soluble receptor molecules

Attachment of a nonaggregating monoclonal antibody and of a soluble recombinant receptor molecule to the icosahedral nonenveloped human rhinovirus serotype 2 was studied with a nanoelectrospray ionization gas-phase electrophoretic molecular mobility analyzer (nESI-GEMMA). The virus mass, as determined via nESI-GEMMA, was within instrument accuracy (+/-6%) close to the theoretical value (8 x 10(6) Da) calculated from the sum of all constituents of one virus particle (60 copies of each of the four viral capsid proteins, the RNA genome, and one copy of the RNA-linked protein VpG). The formation of virus-antibody complexes of different stoichiometries (up to a mass 12.5 x 10(6) Da corresponding to 30 attached antibodies) and virus-receptor complexes (up to a mass 8.8 x 10(6) Da corresponding to 12 attached receptor molecules) was monitored. Via the volume derived from the electrophoretic mobility diameter (EMD), the stoichiometry of the HRV complexes was calculated. The accuracy of the EMD was within +/-0.5 nm, which corresponds to an accuracy of +/-4 antibodies and +/-5 receptor molecules in the respective complexes. For the first time, we here demonstrate the use of nESI-GEMMA for the analysis of the size and stoichiometry of biomolecules in high-order complexes in real time under normal pressure conditions.     

Gradient tandem mass spectrometry interfaced with ion mobility separation for the characterization of supramolecular architectures

Traveling wave ion mobility mass spectrometry (TWIM MS) was combined with gradient tandem mass spectrometry (gMS(2)) to deconvolute and characterize superimposed ions with different charges and shapes formed by electrospray ionization (ESI) of self-assembled, hexameric metallomacrocycles composed of terpyridine-based ligands and Cd(II) ions. ESI conditions were optimized to obtain intact hexameric cation assemblies in a low charge state (2+), in order to minimize overlapping fragments of the same mass-to-charge ratio. With TWIM MS, intact hexameric ions could be separated from remaining fragments and aggregates. Collisional activation of these hexameric ions at varying collision energies (gMS(2)), followed by TWIM separation, was then performed to resolve macrocyclic from linear hexameric species. Because of the different stabilities of these architectures, gMS(2) changes their relative amounts, which can be monitored individually after subsequent ion mobility separation. On the basis of this unique strategy, hexameric cyclic and linear isomers have been successfully resolved and identified. Complementary structural information was gained by the gMS(2) fragmentation pattern of the metallosupramolecules, acquired by collisionally activated dissociation after TWIM dispersion. TWIM MS interfaced with gMS(2) should be particularly valuable for the characterization of a variety of supramolecular polymers, which often contain isomeric architectures that yield overlapping fragments and aggregates upon ESI MS analysis.     

Mass analysis of water-soluble polymers by mobility measurement of charge-reduced ions generated by electrosprays

Aqueous solutions of poly(ethylene glycol) (PEG) in a 10 mM ammonium acetate buffer are electrosprayed, and the maximum charge state on the resulting gas-phase ions is reduced to unity using a radioactive source. The mobility distribution of these charged particles is then measured in air in a differential mobility analyzer of unusually high resolution. The relation Z(m) between the mobility Z of a polymer molecule and its mass m is determined by means of narrowly distributed PEG mass standards. The molecular weight range of available standards is extended by generating clusters containing from one up to six molecules of the primary PEG standard. The mass at the peak of the distribution of the lowest standard (PEG-4k) is determined by MALDI mass spectrometry and agrees with the manufacturer's value and previous MALDI literature data. The masses for the 50K and 120K standards are found to differ by 8.6 and 6.6%, respectively, from the manufacturer's value. Using known relationships, the particle diameter d of the ions is calculated from the measured mobility. Plots of d versus m(1/3) give straight lines over the full mass range studied (4000-700 000 Da, particle diameter from 3 to 12 nm), indicating that these PEG particles are indeed spherical and have a density rho independent of size. The slope of the d versus m(1/3) curve provides a density rho = 1.25 g/cm(3), close to the known bulk density, rho(PEG) = 1.21 g/cm(3).     

A simple and robust method for determining the number of basic sites in peptides and proteins using electrospray ionization mass spectrometry

A solution additive has been discovered that can be used to measure the number of basic sites in a peptide or protein using electrospray ionization (ESI) mass spectrometry. Addition of millimolar amounts of perchloric acid (HClO(4)) to aqueous solutions that contain peptides or proteins results in the noncovalent adduction of HClO(4) molecules to the multiply charged ions formed by ESI. For 18 oligopeptides and proteins, ranging in molecular weight from 0.5 to 18.3 kDa, the sum of the number of protons plus maximum number of HClO(4) molecules adducted to the lower charge state ions is equal to the number of basic sites in the molecule. This method provides a rapid means of obtaining information about the composition of a peptide or protein and does not require high-resolution measurements or any instrumental or experimental modifications.     

Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/ QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/ MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.     

Characterizing the structures and folding of free proteins using 2-D gas-phase separations: observation of multiple unfolded conformers

Understanding the 3-D structure and dynamics of proteins and other biological macromolecules in various environments is among the central challenges of chemistry. Electrospray ionization can often transfer ions from solution to gas phase with only limited structural distortion, allowing their profiling using mass spectrometry and other gas-phase approaches. Ion mobility spectrometry (IMS) can separate and characterize macroion conformations with high sensitivity and speed. However, IMS separation power is generally insufficient for full resolution of major structural variants of protein ions and elucidation of their interconversion dynamics. Here we report characterization of macromolecular conformations using field asymmetric waveform IMS (FAIMS) coupled to conventional IMS in conjunction with mass spectrometry. The collisional heating of ions in the electrodynamic funnel trap between FAIMS and IMS stages enables investigating the structural evolution of particular isomeric precursors as a function of the intensity and duration of activation that can be varied over large ranges. These new capabilities are demonstrated for ubiquitin and cytochrome c, two common model proteins for structure and folding studies. For nearly all charge states, two-dimensional FAIMS/IMS separations distinguish many more conformations than either FAIMS or IMS alone, including some with very low abundance. For cytochrome c in high charge states, we find several abundant "unfolded" isomer series not distinguishable by IMS, possibly corresponding to different "string of beads" geometries. The unfolding of specific ubiquitin conformers selected by FAIMS has been studied by employing their heating in the FAIMS/IMS interface.     

High-resolution field asymmetric waveform ion mobility spectrometry using new planar geometry analyzers

Field asymmetric waveform ion mobility spectrometry (FAIMS) has emerged as a powerful tool of broad utility for separation and characterization of gas-phase ions, especially in conjunction with mass spectrometry (MS). In FAIMS, ions are filtered by the dependence of mobility on electric field while being carried by gas flow through the analytical gap between two electrodes of either planar (p-) or cylindrical (c-) geometry. Most FAIMS/MS systems employ c-FAIMS because of its ease of coupling to MS, yet the merits of the two geometries have not been compared in detail. Here, a priori simulations reveal that reducing the FAIMS curvature always improves resolution at equal sensitivity. In particular, the resolving power of p-FAIMS exceeds that of c-FAIMS, typically by a factor of 2-4 depending on the ion species and carrier gas. We have constructed a new planar FAIMS incorporating a curtain plate interface for effective operation with an ESI ion source and joined to an MS using an ion funnel interface with a novel slit aperture. The resolution increases up to 4-fold over existing c-FAIMS, even though the analysis is approximately 2 times faster. This allows separation of species not feasible in previous FAIMS studies, e.g., protonated leucine and isoleucine or new bradykinin isomers. The improvement for protein conformers (of ubiquitin) is less significant, possibly because of multiple unresolved geometries.     

Supercharged protein and peptide ions formed by electrospray ionization

The multiple charging of large molecules in electrospray ionization provides key advantages for obtaining accurate molecular weights by mass spectrometry and for obtaining structural information by tandem mass spectrometry and MS(n) experiments. Addition of glycerol or m-nitrobenzyl alcohol into the electrospray solutions dramatically increases both the maximum observed charge state and the abundances of the high charge states of protein and peptide ions. Adding glycerol to acidified aqueous solutions of cytochrome c shifts the most abundant charge state from 17+ to 21+, shifts the maximum charge state from 20+ to 23+, and shifts the average charge state from 16.6+ to 20.9+. Much less m-nitrobenzyl alcohol (<1%) is required to produce similar results. With just 0.7% m-nitrobenzyl alcohol, even the 24+ charge state of cytochrome c is readily observed. Similar results are obtained with myoglobin and (Lys)4. For the latter molecule, the 5+ charge state is observed in the electrospray mass spectrum obtained from solutions containing 6.7% m-nitrobenzyl alcohol. This charge state corresponds to protonation of all basic sites in this peptide. Although the mechanism for enhanced charging is unclear, it does not appear to be a consequence of conformational changes of the analyte molecules. This method of producing highly charged protein ions should be useful for improving the performance of mass measurements on mass spectrometers with performances that decrease with increasing m/z. This should also be particularly useful for tandem mass spectrometry experiments, such as electron capture dissociation, for which highly charged ions are desired.     

Compound screening for the presence of the primary N-oxide functionality via ion-molecule reactions in a mass spectrometer

A mass spectrometry method is presented for the identification of compounds that contain the primary N-oxide functional group. This method utilizes a gas-phase ion-molecule reaction with dimethyl disulfide that rapidly and selectively derivatizes the protonated primary N-oxide functional group in a mass spectrometer to yield an ionic reaction product (with 31 Da higher mass than that of the protonated molecule) that is diagnostic for the presence of a primary N-oxide functionality. A variety of protonated analytes containing different functional groups were tested in Fourier transform ion-cyclotron resonance and triple quadrupole mass spectrometers to probe the selectivity of the reaction. Only molecules containing the protonated primary N-oxide functional group yielded the diagnostic reaction product; all other protonated molecules gave protonated dimethyl disulfide or no reaction products. The feasibility of this method for compound screening was tested by examining six analytes with the same molecular formula but different atom connectivity. The one analyte that contained the primary N-oxide functional group was readily differentiated from the other analytes.     

Ambient mass spectrometry with a handheld mass spectrometer at high pressure

The first coupling of atmospheric pressure ionization methods, electrospray ionization (ESI) and desorption electrospray ionization (DESI), to a miniature hand-held mass spectrometer is reported. The instrument employs a rectilinear ion trap (RIT) mass analyzer and is battery-operated, hand-portable, and rugged (total system: 10 kg, 0.014 m(3), 75 W power consumption). The mass spectrometer was fitted with an atmospheric inlet, consisting of a 10 cm x 127 microm inner diameter stainless steel capillary tube which was used to introduce gas into the vacuum chamber at 13 mL/min. The operating pressure was 15 mTorr. Ions, generated by the atmospheric pressure ion source, were directed by the inlet along the axis of the ion trap, entering through an aperture in the dc-biased end plate, which was also operated as an ion gate. ESI and DESI sources were used to generate ions; ESI-MS analysis of an aqueous mixture of drugs yielded detection limits in the low parts-per-billion range. Signal response was linear over more than 3 orders of magnitude. Tandem mass spectrometry experiments were used to identify components of this mixture. ESI was also applied to the analysis of peptides and in this case multiply charged species were observed for compounds of molecular weight up to 1200 Da. Cocaine samples deposited or already present on different surfaces, including currency, were rapidly analyzed in situ by DESI. A geometry-independent version of the DESI ion source was also coupled to the miniature mass spectrometer. These results demonstrate that atmospheric pressure ionization can be implemented on simple portable mass spectrometry systems.     

Coupling capillary electrophoresis and high-field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of complex lipopolysaccharides

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a new technology for atmospheric pressure, room temperature separation of gas-phase ions. The FAIMS system acts as an ion filter that can continuously transmit one type of ion, independent of mass-to-charge ratio (m/z). Capillary electrophoresis-electrospray mass spectrometry (CE-MS) has been extensively used for the analysis of complex bacterial lipopolysaccharides (LPS). The coupling of FAIMS to CE-MS provides a sensitive technique for the characterization of these complex glycolipids, permitting the separation of trace-level LPS oligosaccharide glycoforms for subsequent structural characterization using tandem mass spectrometry. This was demonstrated for LPS from nontypeable Haemophilus influenzae strain 375 following O-deacylation with anhydrous hydrazine. This strain of H. influenzae can express a triheptosyl-containing glycoform to which four hexose residues are linked forming the outer-core region of the molecule. This has been referred to as the Hex4 glycoform. Glycoforms have been identified which differ in the number of phosphoethanolamine substituents in the inner-core. With the use of CE-FAIMS, isomeric Hex4 glycoforms containing two PEtn groups were separated and characterized by MS/MS. FAIMS provided a significant reduction in mass spectral noise, leading to improved detection limits ( approximately 70 amol of the major glycoform). The extracted mass spectrum showed that the apparent noise was virtually eliminated. In addition to the reduction of chemical background, the ion current was increased by as much as 7.5 times as a result of the atmospheric pressure ion-focusing effect provided by the FAIMS system. The linearity of response of the CE-FAIMS-MS system was also studied. The calibration curve is linear for approximately 3 orders of magnitude, over a range of 40 pg/microL to 10 ng/microL.     

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%.     

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.