Simple and Minimally Invasive SID Devices for Native Mass Spectrometry.

We describe a set of simple devices for surface-induced dissociation of proteins and protein complexes on three instrument platforms. All of the devices use a novel yet simple split lens geometry that is minimally invasive (requiring a few millimeters along the ion path axis) and is easier to operate than prior generations of devices. The split lens is designed to be small enough to replace the entrance lens of a Bruker FT-ICR collision cell, the dynamic range enhancement (DRE) lens of a Waters Q-IM-TOF, or the exit lens of a transfer multipole of a Thermo Scientific Extended Mass Range (EMR) Orbitrap. Despite the decrease in size and reduction in number of electrodes to 3 (from 10 to 12 in Gen 1 and ∼6 in Gen 2), we show sensitivity improvement in a variety of cases across all platforms while also maintaining SID capabilities across a wide mass and energy range. The coupling of SID, high resolution, and ion mobility is demonstrated for a variety of protein complexes of varying topologies.

Unique capabilities of AC frequency scanning and its implementation on a Mars Organic Molecule Analyzer linear ion trap.

A limitation of conventional quadrupole ion trap scan modes which use rf amplitude control for mass scanning is that, in order to detect a subset of an ion population, the rest of the ion population must also be interrogated. That is, ions cannot be detected out of order; they must be detected in order of either increasing or decreasing mass-to-charge (m/z). However, an ion trap operated in the ac frequency scan mode, where the rf amplitude is kept constant and instead the ac frequency is used for mass-selective operations, has no such limitation because any variation in the ac frequency affects only the subset of ions whose secular frequencies match the perturbation frequency. Hence, an ion trap operated in the ac frequency scan mode can perform any arbitrary mass scan, as well as a sequence of scans, using a single ion injection; we demonstrate both capabilities here. Combining these two capabilities, we demonstrate the acquisition of a full mass spectrum, a product ion spectrum, and a second generation product ion spectrum using a single ion injection event. We further demonstrate a "segmented scan" in which different mass ranges are interrogated at different rf amplitudes in order to improve resolution over a portion of the mass range, and a "periodic scan" in which ions are continuously introduced into the ion trap to achieve a nearly 100% duty cycle. These unique scan modes, along with other characteristics of ac frequency scanning, are particularly appropriate for miniature ion trap mass spectrometers. Hence, implementation of ac frequency scanning on a prototype of the Mars Organic Molecule Analyzer mass spectrometer is also described.

Improved Differential Ion Mobility Separations Using Linked Scans of Carrier Gas Composition and Compensation Field.

Differential ion mobility spectrometry (DIMS) separates ions based on differences in their mobilities in low and high electric fields. When coupled to mass spectrometric analyses, DIMS has the ability to improve signal-to-background by eliminating isobaric and isomeric compounds for analytes in complex mixtures. DIMS separation power, often measured by resolution and peak capacity, can be improved through increasing the fraction of helium in the nitrogen carrier gas. However, because the mobility of ions is higher in helium, a greater number of ions collide with the DIMS electrodes or housing, yielding losses in signal intensity. To take advantage of the benefits of helium addition on DIMS separations and reduce ion losses, linked scans were developed. In a linked scan the helium content of the carrier gas is reduced as the compensation field is increased. Linked scans were compared with conventional compensation field scans with constant helium content for the protein ubiquitin and a tryptic digest of bovine serum albumin (BSA). Linked scans yield better separation of ubiquitin charge states and enhanced peak capacities for the analysis of BSA compared with compensation field scans with constant helium carrier gas percentages. Linked scans also offer improved signal intensity retention in comparison to compensation field scans with constant helium percentages in the carrier gas.

Ion dynamics in a trapped ion mobility spectrometer.

In the present paper, theoretical simulations and experimental observations are used to describe the ion dynamics in a trapped ion mobility spectrometer. In particular, the ion motion, ion transmission and mobility separation are discussed as a function of the bath gas velocity, radial confinement, analysis time and speed. Mobility analysis and calibration procedure are reported for the case of sphere-like molecules for positive and negative ion modes. Results showed that a maximal mobility resolution can be achieved by optimizing the gas velocity, radial confinement (RF amplitude) and ramp speed (voltage range and ramp time). The mobility resolution scales with the electric field and gas velocity and R = 100-250 can be routinely obtained at room temperature.

High-field asymmetric-waveform ion mobility spectrometry and electron detachment dissociation of isobaric mixtures of glycosaminoglycans.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is shown to be capable of resolving isomeric and isobaric glycosaminoglycan negative ions and to have great utility for the analysis of this class of molecules when combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tandem mass spectrometry. Electron detachment dissociation (EDD) and other ion activation methods for tandem mass spectrometry can be used to determine the sites of labile sulfate modifications and for assigning the stereochemistry of hexuronic acid residues of glycosaminoglycans (GAGs). However, mixtures with overlapping mass-to-charge values present a challenge, as their precursor species cannot be resolved by a mass analyzer prior to ion activation. FAIMS is shown to resolve two types of mass-to-charge overlaps. A mixture of chondroitin sulfate A (CSA) oligomers with 4-10 saccharides units produces ions of a single mass-to-charge by electrospray ionization, as the charge state increases in direct proportion to the degree of polymerization for these sulfated carbohydrates. FAIMS is shown to resolve the overlapping charge. A more challenging type of mass-to-charge overlap occurs for mixtures of diastereomers. FAIMS is shown to separate two sets of epimeric GAG tetramers. For the epimer pairs, the complexity of the separation is reduced when the reducing end is alkylated, suggesting that anomers are also resolved by FAIMS. The resolved components were activated by EDD and the fragment ions were analyzed by FTICR-MS. The resulting tandem mass spectra were able to distinguish the two epimers from each other.

Activated ion negative electron transfer dissociation of multiply charged peptide anions.

We report the implementation and evaluation of activated ion negative electron transfer dissociation (AI-NETD) in order to enhance the analytical capabilities of NETD for the elucidation of doubly deprotonated peptide anions. The analytical figures-of-merit and fragmentation characteristics are compared for NETD alone and with supplemental collisional activation of the charge reduced precursors or infrared photoactivation of the entire ion population during the NETD reaction period. The addition of supplemental collisional activation of charge reduced precursor ions or infrared photoactivation of the entire ion population concomitant with the NETD reaction period significantly improves sequencing capabilities for peptide anions as evidenced by the greater abundances of product ions and overall sequence coverage. Neither of these two AI-NETD methods significantly alters the net fragmentation efficiencies relative to NETD; however, the sequence ion conversion percentages with respect to formation of diagnostic product ions are notably higher. Supplemental infrared photoactivation outperforms collisional activation for most of the peptide fragmentation metrics evaluated.

Gas-phase separation using a trapped ion mobility spectrometer.

In the present work we describe the principles of operation, versatility and applicability of a trapped ion mobility spectrometer (TIMS) analyzer for fast, gas-phase separation of molecular ions based on their size-to-charge ratio. Mobility-based separation using a TIMS device is shown for a series for isobar pairs. In a TIMS device, mobility resolution depends on the bath gas velocity and analysis scan speed, with the particularity that the mobility separation can be easily tuned from low to high resolution (R>50) in accordance with the analytical challenge. In contrast to traditional drift tube IMS analyzer, a TIMS device can be easily integrated in a mass spectrometer without a noticeable loss in ion transmission or sensitivity, thus providing a powerful separation platform prior to mass analysis.

Negative electron transfer dissociation of glycosaminoglycans.

Structural characterization of glycosaminoglycans (GAGs) has been a challenge in the field of mass spectrometry, and the application of electron detachment dissociation (EDD) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has shown great promise to GAG oligosaccharide characterization in a single tandem mass spectrometry experiment. In this work, we apply the technique of negative electron transfer dissociation (NETD) to GAGs on a commercial ion trap mass spectrometer. NETD of GAGs, using fluoranthene or xenon as the reagent gas, produces fragmentation very similar to previously observed EDD fragmentation. Using fluoranthene or xenon, both glycosidic and cross-ring cleavages are observed, as well as even- and odd-electron products. The loss of SO(3) can be minimized and an increase in cross-ring cleavages is observed if a negatively charged carboxylate is present during NETD, which can be controlled by the charge state or the addition of sodium. NETD effectively dissociates GAGs up to eight saccharides in length, but the low resolution of the ion trap makes assigning product ions difficult. Similar to EDD, NETD is also able to distinguish the epimers iduronic acid from glucuronic acid in heparan sulfate tetrasaccharides and suggests that a radical intermediate plays an important role in distinguishing these epimers. These results demonstrate that NETD is effective at characterizing GAG oligosaccharides in a single tandem mass spectrometry experiment on a widely available mass spectrometry platform.

Negative electron transfer dissociation of deprotonated phosphopeptide anions: choice of radical cation reagent and competition between electron and proton transfer.

Despite significant developments in mass spectrometry technology in recent years, no routine proteomics sequencing tool is currently available for peptide anions. The use of a molecular open-shell cation is presented here as a possible reaction partner to induce electron transfer dissociation with deprotonated peptide anions. In this negative electron transfer dissociation (NETD) scheme, an electron is abstracted from the peptide anion and transferred to the radical cation. This is demonstrated for the example of the fluoranthene cation, C(16)H(10)(+*), which is reacted with deprotonated phosphorylated peptides in a 3-D ion trap mass spectrometer. Selective backbone cleavage at the C(alpha)-C bond is observed to yield a and x fragments, similarly to electron detachment dissociation (EDD) of peptide anions. Crucially, the phosphorylation site is left intact in the dissociation process, allowing an identification and localization of the post-translational modification (PTM) site. In contrast, NETD using Xe(+*) as the reagent cation results in sequential neutral losses (CO(2) and H(3)PO(4)) from a/x fragments, which complicate the interpretation of the mass spectra. This difference in dissociation behavior can be understood in the framework of the reduced recombination energy of the electron transfer process for fluoranthene, which is estimated at 2.5-4.5 eV, compared to 6.7-8.7 eV for xenon. Similarly to ETD, proton transfer is found to compete with electron transfer processes in NETD. Isotope fitting of the charge-reduced species shows that in the case of fluoranthene-mediated NETD, proton transfer only accounts for <20%, whereas this process highly abundant for Xe(+*) (43 and 82%). Since proton abstraction from Xe(+*) is not possible, this suggests that Xe(+*) ionizes other transient species in the ion trap, which then engage in proton transfer reactions with the peptide anions.

Data-dependent electron transfer dissociation of large peptides and medium size proteins in a QTOF instrument on a liquid chromatography timescale.

Liquid chromatography (LC) electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) of protein digests is demonstrated in a hybrid quadrupole-hexapole orthogonal time-of-flight (OTOF) mass spectrometer. Analyte ions are selected in a mass-analyzing quadrupole, accumulated in the hexapole linear ETD reaction cell and mutually stored with ETD reagent anions. Product ions are collected in an ion cooler and then analyzed by an OTOF mass analyzer. The hexapole structure of the ETD reaction cell allows for a broad fragment ion mass range distribution and a high ion storage capacity. Analytically useful ETD OTOF-MS/MS spectra could be obtained at a rate of faster than 2 Hz. When used in conjunction with LC this high speed allows for several MS and MS/MS spectra to be obtained across each LC peak. An MS scan is used to select the precursor ions. With a 1 m flight tube and single reflection, resolutions of about 10 k and a mass accuracy of 5 ppm were achieved. When analyzing a 100 fmol solution of a tryptic digest of bovine serum albumin (BSA) by LC/ETD MS/MS, 27 unique peptides were identified with a summed Mascot score of 1316 using the Swiss Prot database. In addition, we explored the capability for analyzing small proteins with the present hybrid instrument. ETD MS/MS of intact ubiquitin ([M+12H](12+)) leads to the identification of the protein with a Mascot score of 264.

Protein conformations can be probed in top-down HDX MS experiments utilizing electron transfer dissociation of protein ions without hydrogen scrambling.

Electron-transfer dissociation (ETD) is evaluated as a technique to provide local information on higher order structure and dynamics of a whole protein molecule. Isotopic labeling of highly flexible segments of a model 18 kDa protein is carried out in solution under mildly denaturing conditions by means of hydrogen/deuterium exchange (HDX), followed by transfer of intact protein ions to the gas phase by means of electrospray ionization, and mass-selection of a precursor ion for subsequent reactions with fluoranthene radical anions. The ETD process gives rise to abundant fragment ions, whose deuterium content can be measured as a function of duration of the HDX reaction in solution. No backbone protection is detected for all protein segments spanning the 25-residue long N-terminal part of the protein, which is known to lack structure in solution. At the same time, noticeable protection is evident for segments representing the structured regions of the protein. The results of this work suggest that ETD of intact protein ions is not accompanied by detectable hydrogen scrambling and can be used in tandem with HDX to probe protein conformation in solution.

Pulsed Nano-Electrospray Ionization: Characterization of Temporal Response and Implementation with a Flared Inlet Capillary.

The temporal response of pulsed nano-electrospray ionization mass spectrometry (nano-ESI-MS) was studied and its influence on ion formation and detection was characterized. Rise and decay times for the mass resolved ion current were determined to be 20 ± 3 msec and 61 ± 5 msec, respectively, which led to a maximum pulse rate of 12 Hz. Pulsed nano-ESI operation was demonstrated from a multi-sprayer source controlled by a high voltage pulsing circuit constructed in-house. The desired source mode of operation (e.g. pulsing or continuous) can be realized solely by controlling the voltage applied to each sprayer.

Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer.

Electron transfer dissociation (ETD) of proteins is demonstrated in a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer (Qh-FTICRMS). Analyte ions are selected in the mass analyzing quadrupole, accumulated in the hexapole linear ion trap, reacted with fluoranthene reagent anions, and then analyzed via an FTICR mass analyzer. The hexapole trap allows for a broad fragment ion mass range and a high ion storage capacity. Using a 3 T FTICRMS, resolutions of 60 000 were achieved with mass accuracies averaging below 1.4 ppm. The high resolution, high mass accuracy ETD spectra provided by FTICR obviates the need for proton transfer reaction (PTR) charge state reduction of ETD product ions when analyzing proteins or large peptides. This is demonstrated with the ETD of ubiquitin and apomyoglobin yielding sequence coverages of 37 and 20%, respectively. We believe this represents the first reported successful combination of ETD and a FTICRMS.

Improved mass accuracy for higher mass peptides by using SWIFT excitation for MALDI-FTICR mass spectrometry.

Stepwise-external calibration has previously been shown to produce sub part-per-million (ppm) mass accuracy for the MALDI-FTICR/MS analyses of peptides up to m/z 2500. The present work extends these results to ions up to m/z 4000. Mass measurement errors for ions of higher mass-to-charge are larger than for ions below m/z 2500 when using conventional chirp excitation to detect ions. Mass accuracy obtained by using stored waveform inverse Fourier transform (SWIFT) excitation was evaluated and compared with chirp excitation. Analysis of measurement errors reveals that SWIFT excitation provides smaller deviations from the calibration equation and better mass accuracy than chirp excitation for a wide mass range and for widely varying ion populations.