Time-of-flight mass spectrometry with an electrospray ion beam.

An electrospray ionization source has been coupled to a reflection time-of-flight mass spectrometer. By orienting the ion source perpendicular to the field-free drift region, the longitudinal energy spread of the ion packet has been substantially reduced, allowing a mass resolving power of over 1000 to be achieved for both low-mass and high-mass ions of biological interest. In addition, instrument sensitivity allows the routine detection of low-picomole and subpicomole quantities of large multiply charged species such as cytochrome c (MW = 12,360.9). The potential utility of this instrument for conducting rapid screening of chromatographic effluents is discussed in light of its simplicity, rapid scanning speed, and high sensitivity.     

A quadrupole ion trap mass spectrometer with three independent ion sources for the study of gas-phase ion/ion reactions

An instrument for the study of gas-phase ion/ion reactions in which three independent sources of ions, namely, two electrospray ionization sources and one atmospheric sampling glow discharge ionization source, are interfaced to a quadrupole ion trap mass analyzer is described. This instrument expands the scope of gas-phase ion/ion reaction studies by allowing for manipulation of the charge states of multiply charged reactant and product ions. Examples are provided involving the formation of protein-protein complexes in the gas phase. Complexes with charge states that cannot be formed from reactant ion charge states present in the normal electrospray charge state distributions can be formed in the new apparatus. Strategies that rely on both reactant ion charge state manipulation and product ion charge state manipulation are demonstrated. In addition, simplification of product ion spectra generated from dissociation of complexes formed via ion/ion reactions can be effected by using the discharge source to reduce the charge state of the product ions to primarily 1+.     

Development and characterization of an ionization technique for analysis of biological macromolecules: liquid matrix-assisted laser desorption electrospray ionization

We have developed an atmospheric pressure ionization technique called liquid matrix-assisted laser desorption electrospray ionization (liq-MALDESI) for the generation of multiply charged ions by laser desorption from liquid samples deposited onto a stainless steel sample target biased at a high potential. This variant of our previously reported MALDESI source does not utilize an ESI emitter to postionize neutrals. Conversely, we report desorption and ionization from a macroscopic charged droplet. We demonstrate high mass resolving power single-acquisition FT-ICR-MS analysis of peptides and proteins ranging from 1 to 8.6 kDa at atmospheric pressure. The liquid sample acts as a macroscopic charged droplet similar to those generated by electrospray ionization, whereby laser irradiation desorbs analyte from organic matrix containing charged droplets generating multiply charged ions. We have observed a singly charged radical cation of an electrochemically active species indicating oxidation occurs for analytes and therefore water; the latter would play a key role in the mechanism of ionization. Moreover, we demonstrate an increase in ion abundance and a concurrent decrease in surface tension with an increase in the applied potential.     

New paradigm in ionization: multiply charged ion formation from a solid matrix without a laser or voltage

Laserspray ionization (LSI) is a new approach to producing multiply charged ions from solids on surfaces by laser ablation of matrixes commonly used in matrix-assisted laser desorption/ionization (MALDI). We show that the only necessity of the laser for producing multiply charged ions is to deliver particles or droplets of the matrix/analyte mixture to an ionization zone which is simply a heated inlet to the vacuum of the mass spectrometer. Several other methods for delivering sample are demonstrated to produce nearly equivalent results. One example shows the use of an air gun replacing the laser and producing mass spectra of proteins by shooting pellets into a metal plate which has matrix/analyte applied to the opposite side and near the ion entrance inlet to the mass spectrometer. Multiply charged ions of proteins are produced in the absence of any electric field or laser and with only the need of a heated ion entrance capillary or skimmer. The commonality of the matrix with MALDI and the mild conditions necessary for formation of ions brings into question the mechanism of formation of multiply charged ions and the importance of matrix structure in this process.     

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%.     

Commercial intermediate pressure MALDI ion mobility spectrometry mass spectrometer capable of producing highly charged laserspray ionization ions

The first examples of highly charged ions observed under intermediate pressure (IP) vacuum conditions are reported using laser ablation of matrix/analyte mixtures. The method and results are similar to those obtained at atmospheric pressure (AP) using laserspray ionization (LSI) and/or matrix assisted inlet ionization (MAII). Electrospray ionization (ESI), LSI, and MAII are methods operating at AP and have been shown, with or without the use of a voltage or a laser, to produce highly charged ions with very similar ion abundance and charge states. A commercial matrix-assisted laser desorption/ionization ion mobility spectrometry (IMS) mass spectrometry (MS) instrument (SYNAPT G2) was used for the IP developments. The necessary conditions for producing highly charged ions of peptides and small proteins at IP appear to be a pressure drop region and the use of suitable matrixes and laser fluence. Ionization to produce these highly charged ions under the low pressure conditions of IP does not require specific heating or a special inlet ion transfer region. However, under the current setup, ubiquitin is the highest molecular weight protein observed. These findings are in accord with the need to provide thermal energy in the pressure drop region, similar to LSI and MAII, to improve sensitivity and extend the types of compounds that produce highly charged ions. The practical utility of IP-LSI in combination with IMS-MS is demonstrated for the analysis of model mixtures composed of a lipid, peptides, and a protein. Further, endogenous multiply charged peptides are observed directly from delipified mouse brain tissue with drift time distributions that are nearly identical in appearance to those obtained from a synthesized neuropeptide standard analyzed by either LSI- or ESI-IMS-MS at AP. Efficient solvent-free gas-phase separation enabled by the IMS dimension separates the multiply charged peptides from lipids that remained on the delipified tissue. Lipid and peptide families are exceptionally well separated because of the ability of IP-LSI to produce multiple charging.     

Alternately pulsed nanoelectrospray ionization/atmospheric pressure chemical ionization for ion/ion reactions in an electrodynamic ion trap

The alternate operation of nanoelectrospray ionization and atmospheric pressure chemical ionization, using a common atmosphere/vacuum interface and ion path, has been implemented to facilitate ion/ion reaction experiments in a linear ion trap-based tandem mass spectrometer. The ion sources are operated in opposite polarity modes whereby one of the ion sources is used to form analyte ions while the other is used to form reagent ions of opposite polarity. This combination of ion sources is well-suited to implementation of experiments involving multiply charged ions in reaction with singly charged ions of opposite polarity. Three analytically useful ion/ion reaction types are illustrated: the partial deprotonation of a multiply protonated protein, the partial protonation of a multiply deprotonated oligonucleotide, and electron transfer to a multiply protonated peptide. The approach described herein is attractive in that it enables both single proton-transfer and single electron-transfer ion/ion reaction experiments to be implemented without requiring major modifications to the tandem mass spectrometer hardware. Furthermore, a wide range of reactant ions can be formed with these ionization methods and the pulsed nature of operation appears to lead to no significant compromise in the performance of either ion source.     

Radiative ion-ion neutralization: a new gas-phase atmospheric pressure ion transduction mechanism

All atmospheric pressure ion detectors, including photo ionization detectors, flame ionization detectors, electron capture detectors, and ion mobility spectrometers, utilize Faraday plate designs in which ionic charge is collected and amplified. The sensitivity of these Faraday plate ion detectors are limited by thermal (Johnson) noise in the associated electronics. Thus approximately 10(6) ions per second are required for a minimal detection. This is not the case for ion detection under vacuum conditions where secondary electron multipliers (SEMs) can be used. SEMs produce a cascade of approximately 10(6) electrons per ion impinging on the conversion dynode. Similarly, photomultiplier tubes (PMTs) can generate approximately 10(6) electrons per photon. Unlike SEMs, however, PMTs are evacuated and sealed so that they are commonly used under atmospheric pressure conditions. This paper describes an atmospheric pressure ion detector based on coupling a PMT with light emitted from ion-ion neutralization reactions. The normal Faraday plate collector electrode was replaced with an electrode "needle" used to concentrate the anions as they were drawn to the tip of the needle by a strong focusing electric field. Light was emitted near the surface of the electrode when analyte ions were neutralized with cations produced from the anode. Although radiative-ion-ion recombination has been previously reported, this is the first time ions from separate ionization sources have been combined to produce light. The light from this radiative-ion-ion-neutralization (RIIN) was detected using a photon multiplier such that an ion mobility spectrum was obtained by monitoring the light emitted from mobility separated ions. An IMS spectrum of nitroglycerin (NG) was obtained utilizing RIIN for tranducing the mobility separated ions into an analytical signal. The implications of this novel ion transduction method are the potential for counting ions at atmospheric pressure and for obtaining ion specific emission spectra for mobility separated ions.     

Necessary conditions for peak resolution in an ion mobility increment spectrometer with a flat drift chamber

New analytical characteristics are proposed to describe the performance of an ion mobility increment spectrometer (IMIS). The dispersion describes the ability of such a spectrometer to separate ions with close values of the variable component of mobility. The necessary resolution limit with respect to this component characterizes the ability to separately detect different ion species. In accordance with the principle of ion separation in this instrument, it is suggested to redefine the resolving power of IMIS as the inverse of the necessary resolution limit with respect to the variable component of mobility.     

Implementation of ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer

A commercial quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been adapted for ion/ion reaction studies. To enable mutual storage of oppositely charged ions in a linear ion trap, the oscillating quadrupole field of the second quadrupole of the system (Q2) serves to store ions in the radial dimension while auxiliary radio frequency is superposed on the end lenses of Q2 during the reaction period to create barriers in the axial dimension. A pulsed dual electrospray (ESI) source is directly coupled to the instrument interface for the purpose of proton transfer reactions. Singly and doubly charged protein ions as high in mass as 66 kDa are readily formed and observed after proton-transfer reactions. For the modified instrument, the mass resolving power is approximately 8000 for a wide m/z range, and the mass accuracy is approximately 20 ppm for external calibration and approximately 5 ppm for internal calibration after ion/ion reactions. Parallel ion parking is demonstrated with a six-component protein mixture, which shows the potential application of reducing spectral complexity and concentrating certain charge states. The current system has high flexibility with respect to defining MS(n) experiments involving collision-induced dissociation (CID) and ion/ion reactions. Protein precursor and CID product masses can be determined with good accuracy, providing an attractive platform for top-down proteomics. Electron transfer dissociation ion/ion reactions are implemented by using a pulsed nano-ESI/atmospheric pressure chemical ionization dual source for ionization. The reaction between protonated peptide ions and radical anions of 1,3-dinitrobenzene formed exclusively c- and z-type fragment ions.     

Resolving structural isomers of monosaccharide methyl glycosides using drift tube and traveling wave ion mobility mass spectrometry

Monosaccharide structural isomers including sixteen methyl-D-glycopyranosides and four methyl-N-acetylhexosamines were subjected to ion mobility measurements by electrospray ion mobility mass spectrometry. Two ion mobility-MS systems were employed: atmospheric pressure drift tube ion mobility time-of-flight mass spectrometry and a Synapt G2 HDMS system which incorporates a low pressure traveling wave ion mobility separator. All the compounds were investigated as [M + Na](+) ions in the positive mode. A majority of the monosaccharide structural isomers exhibited different mobility drift times in either system, depending on differences in their anomeric and stereochemical configurations. In general, drift time patterns (relative drift times of isomers) matched between the two instruments. Higher resolving power was observed using the atmospheric pressure drift tube. Collision cross section values of monosaccharide structural isomers were directly calculated from the atmospheric pressure ion mobility experiments, and a collision cross section calibration curve was made for the traveling wave ion mobility instrument. Overall, it was demonstrated that ion mobility-mass spectrometry using either drift tube or traveling wave ion mobility is a valuable technique for resolving subtle variations in stereochemistry among the sodium adducts of monosaccharide methyl glycosides.     

Interfacing an ion mobility spectrometry based explosive trace detector to a triple quadrupole mass spectrometer

Hardware from a commercial-off-the-shelf (COTS) ion mobility spectrometry (IMS) based explosive trace detector (ETD) has been interfaced to an AB/SCIEX API 2000 triple quadrupole mass spectrometer. To interface the COTS IMS based ETD to the API 2000, the faraday plate of the IMS instrument and the curtain plate of the mass spectrometer were removed from their respective systems and replaced by a custom faraday plate, which was fabricated with a hole for passing the ion beam to the mass spectrometer, and a custom interface flange, which was designed to attach the IMS instrument onto the mass spectrometer. Additionally, the mass spectrometer was modified to increase the electric field strength and decrease the pressure in the differentially pumped interface, causing a decrease in the effect of collisional focusing and permitting a mobility spectrum to be measured using the mass spectrometer. The utility of the COTS-ETD/API 2000 configuration for the characterization of the gas phase ion chemistry of COTS-ETD equipment was established by obtaining mass and tandem mass spectra in the continuous ion flow and selected mobility monitoring operating modes and by obtaining mass-selected ion mobility spectra for the explosive standard 2,4,6 trinitrotoluene (TNT). This analysis confirmed that the product ion for TNT is [TNT - H](-), the predominant collision-induced dissociation pathway for [TNT- H](-) is the loss of NO and NO(2), and the reduced mobility value for [TNT - H](-) is 1.54 cm(2)V(-1) s(-1). Moreover, this analysis was attained for sample amounts of 1 ng and with a resolving power of 37. The objective of the research is to advance the operational effectiveness of COTS IMS based ETD equipment by developing a platform that can facilitate the understanding of the ion chemistry intrinsic to the equipment.     

Tandem mass spectrometry of very large molecules: serum albumin sequence information from multiply charged ions formed by electrospray ionization.

Serum albumin proteins, Mr approximately 66 kDa, from 10 different species (bovine, human, rat, horse, sheep, goat, rabbit, dog, porcine, and guinea pig) have been studied by electrospray ionization mass spectrometry (ESI-MS) and tandem MS using a triple-quadrupole instrument. The effectiveness of collisional activation for the multiply charged albumin ions greatly exceeds that for singly charged ions, allowing an extension by a factor of at least 20 to the molecular mass range for obtaining sequence-specific product ions by tandem MS. Efficient dissociation is largely attributed to "preheating" in the interface Coulombic instability and the large number of collisions. Increasing the electric field in the intermediate pressure region, between the nozzle-skimmer elements of the atmospheric pressure/vacuum interface, allows fragmentation of the multiply protonated (to 96+) molecules produced by ESI. The most abundant dissociation product ions assigned have a low charge state (2+ to 5+) and are attributed to "bn" mode species from cleavage of the -CO-N- peptide backbone bonds. Particularly abundant dissociation products originate from regions near residues n = 20-25 from the NH2 terminus for parent ions of moderate charge (approximately 50+). Collisionally activated dissociation (CAD) mass spectra from porcine serum albumin, in contrast to the other albumins, also gave prominent singly charged "yn" fragments formed from cleavages near the COOH terminus. Tandem mass spectrometry (MS/MS) of the multiply charged molecular ions, and of fragment species produced by dissociation in the interface (i.e., effective MS/MS/MS), produced similar "bn" species and served to confirm spectral assignments. We also show that ESI mass spectra allow a qualitative assessment of protein microheterogeneity and, in some cases, resolution of major contributions. The physical and analytical implications of the results are discussed, including the identification of possible errors in previously published sequences.     

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.     

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.     

Use of a single-quadrupole mass spectrometer for collision-induced dissociation studies of multiply charged peptide ions produced by electrospray ionization.

The feasibility of obtaining the collision-induced dissociation (CID) spectra of multiply charged peptide ions produced by electrospray ionization in a simple and inexpensive single-quadrupole mass spectrometer is demonstrated. Collisional activation was carried out in the high-pressure region between the capillary exit and the skimmer entrance to the mass analyzer. The CID of multiply charged peptide ions is very efficient, and the observed fragment ion intensities are typically 1-5% of the parent ion intensity prior to CID. About 70 pmol of the peptide is consumed in obtaining each CID spectrum. Spectra obtained by CID of multiply charged ions from bradykinin, angiotensin II, two peptides with features similar to tryptic peptides, and a synthetic analogue of a component of TGF-alpha containing two disulfide bonds are shown. The influence of the primary structure of the peptide on the observed fragmentation pathways is discussed. Although the present single-quadrupole configuration is simple and effective, the inability to choose a particular parent ion for collisional activation makes it less powerful than the triple-quadrupole configuration for mixtures of peptides and peptide samples that yield more than one charge state in the normal mass spectrum. However, it has the potential for inexpensively obtaining sequence information of proteins at high sensitivity by analyzing the pure tryptic peptides obtained by on-line or off-line chromatographic separation of tryptic digests.     

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry combined with mass spectrometry for peptide analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection.     

Charge ratio analysis method: approach for the deconvolution of electrospray mass spectra

A new method to interpret electrospray mass spectral data based on calculating the ratio of mass-to-charge (m/z) values of multiply charged ions is described. The mass-to-charge ratios of any two multiply charged ions corresponding to a single compound are unique numbers that enable the charge states for each ion to be unequivocally identified. The multiply charged ions in electrospray mass spectra originate from the addition or abstraction of protons, cations, or anions to and from a compound under analysis. In contrast to existing deconvolution processes, the charge ratio analysis method (CRAM), identifies the charge states of multiply charged ions without any prior knowledge of the nature of the charge-carrying species. In the case of high-resolution electrospray mass spectral data, in which multiply charged ions are resolved to their isotopic components, the CRAM is capable of correlating the isotope peaks of different multiply charged ions that share the same isotopic composition. This relative ratio method is illustrated here for electrospray mass spectral data of lysozyme and oxidized ubiquitin recorded at low- to high-mass resolution on quadrupole ion trap and Fourier transform ion cyclotron mass spectrometers, and theoretical data for the protein calmodulin based upon a reported spectrum recorded on the latter.     

Ultrahigh-resolution differential ion mobility spectrometry using extended separation times

Ion mobility spectrometry (IMS), and particularly differential IMS or field asymmetric waveform IMS (FAIMS), is emerging as a versatile tool for separation and identification of gas-phase ions, especially in conjunction with mass spectrometry. For over two decades since its inception, the utility of FAIMS was constrained by resolving power (R) of less than ～20. Stronger electric fields and optimized gas mixtures have recently raised achievable R to ～200, but further progress with such approaches is impeded by electrical breakdown. However, the resolving power of planar FAIMS devices using any gas and field intensity scales as the square root of separation time (t). Here, we extended t from the previous maximum of 0.2 s up to 4-fold by reducing the carrier gas flow and increased the resolving power by up to 2-fold as predicted, to >300 for multiply charged peptides. The resulting resolution gain has enabled separation of previously "co-eluting" peptide isomers, including folding conformers and localization variants of modified peptides. More broadly, a peak capacity of ～200 has been reached in tryptic digest separations.     

Extending the laserspray ionization concept to produce highly charged ions at high vacuum on a time-of-flight mass analyzer

A new matrix compound, 2-nitrophloroglucinol, is reported which not only produces highly charged ions similar to electrospray ionization (ESI) under atmospheric pressure (AP) and intermediate pressure (IP) laserspray ionization (LSI) conditions but also the most highly charged ions so far observed for small proteins in mass spectrometry (MS) under high vacuum (HV) conditions. This new matrix extends the compounds that can successfully be employed as matrixes with LSI, as demonstrated on an LTQ Velos (Thermo) at AP, a matrix-assisted laser desorption/ionization (MALDI)-ion mobility spectrometry (IMS) time-of-flight (TOF) SYNAPT G2 (Waters) at IP, and MALDI-TOF Ultraflex, UltrafleXtreme, and Autoflex Speed (Bruker) mass spectrometers at HV. Measurements show that stable multiple charged molecular ions of proteins are formed under all pressure conditions indicating softer ionization than MALDI, which suffers a high degree of metastable fragmentation when multiply charged ions are produced. An important analytical advantage of this new LSI matrix are the potential for high sensitivity equivalent or better than AP-LSI and vacuum MALDI and the potential for enhanced mass selected fragmentation of the abundant highly charged protein ions. A second new LSI matrix, 4,6-dinitropyrogallol, produces abundant multiply charged ions at AP but not under HV conditions. The differences in these similar compounds ability to produce multiply charged ions under HV conditions is believed to be related to their relative ability to evaporate from charged matrix/analyte clusters.