Protonated Leucine Enkephalin Research Articles

Effects of Nano-Electrospray Ionization Emitter Position on Unintentional In-Source Activation of Peptide and Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-electrospray ionization (nESI). Many nESI setups have mobile stages for tuning the ion signal and extent of co-solute and salt adduction. However, tuning the position of the emitter capillary in nESI can have unintended downstream consequences for collision-induced unfolding or collision-induced dissociation (CIU/D) experiments. Here, we show that relatively small variations in the nESI emitter position can shift the midpoint (commonly called the "CID50" or "CIU50") potential of CID breakdown curves and CIU transitions by as much as 8 V on commercial instruments. A spatial "map" of the shift in CID50 for the loss of heme from holomyoglobin onto the emitter position on a Waters Synapt G2-Si mass spectrometer shows that emitter positions closer to the instrument inlet can result in significantly greater in-source activation, whereas different effects are found on an Agilent 6545XT instrument for the ions studied. A similar effect is observed for CID of the singly protonated leucine enkephalin peptide and Shiga toxin 1 subunit B homopentamer on the Waters Synapt G2-Si instrument. In-source activation effects on a Waters Synapt G2-Si are also investigated by examining the RMSD between CIU fingerprints acquired at different emitter positions and the shifts in CIU50 for structural transitions of bovine serum albumin and NIST monoclonal antibody.

Dissociation Kinetics in Quadrupole Ion Traps: Effective Temperatures under Dipolar DC Collisional Activation Conditions.

Ions stored in an electrodynamic ion trap can be forced from the center of the ion trap to regions of higher radio frequency (RF) electric fields by exposing them to a dipolar DC (DDC) potential applied across opposing electrodes. Such ions absorb power from the trapping RF field, resulting in increased ripple motion at the frequency of the trapping RF. When a bath gas is present, ions undergo energetic collisions that result in "RF-heating" sufficient to induce fragmentation. DDC is therefore a broad-band (i.e., mass-to-charge-independent) means for collisional activation in ion traps with added bath gas. Under appropriate conditions, the internal energy distribution of an ion population undergoing dissociation can be approximated with an effective temperature, Teff. In such cases, it is possible to determine thermal activation parameters, such as Arrhenius activation energies and A-factors, by measuring dissociation kinetics. In this work, the well-studied thermometer ion, protonated leucine enkephalin, was subjected to DDC activation under rapid energy exchange conditions and in two separate bath gases, N2 and Ar, to measure Teff as a function of the ratio of DDC and RF voltages. As a result, an empirically derived calibration was generated to link experimental conditions to Teff. It was also possible to quantitatively evaluate a model described by Tolmachev et al. that can be used to predict Teff. It was found that the model, which was derived under the assumption of an atomic bath gas, accurately predicts Teff when Ar was used as the bath gas but overestimates Teff when N2 was the bath gas. Adjustment of the Tolmachev et al. model for a diatomic gas resulted in an underestimate of Teff. Thus, use of an atomic gas can provide accurate activation parameters, while an empirical correction factor should be used to generate activation parameters using N2.

Design and Performance of a Second-Generation Surface-Induced Dissociation Cell for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Native Protein Complexes.

A second-generation ("Gen 2") device capable of surface-induced dissociation (SID) and collision-induced dissociation (CID) for Fourier transform ion cyclotron resonance mass spectrometry of protein complexes has been designed, simulated, fabricated, and experimentally compared to a first-generation device ("Gen 1"). The primary goals of the redesign were to (1) simplify SID by reducing the number of electrodes, (2) increase CID and SID sensitivity by lengthening the collision cell, and (3) increase the mass range of the device for analysis of larger multimeric proteins, all while maintaining the normal instrument configuration and operation. Compared to Gen 1, Gen 2 exhibits an approximately 10× increase in sensitivity in flythrough mode, 7× increase in CID sensitivity for protonated leucine enkephalin (m/z 556), and 14× increase of CID sensitivity of 53 kDa streptavidin tetramer. It also approximately doubles the useful mass range (from m/z 8000 to m/z 15 000) using a rectilinear ion trap with a smaller inscribed radius or triples it (to m/z 22 000) using a hexapole collision cell and yields a 3-10× increase in SID sensitivity. We demonstrate the increased mass range and sensitivity on a variety of model molecules spanning nearly 3 orders of magnitude in absolute mass and present examples where the high resolution of the FT-ICR is advantageous for deconvoluting overlapping SID fragments.

Enhancement of Ion Activation and Collision-Induced Dissociation by Simultaneous Dipolar Excitation of Ions in x- and y-Directions in a Linear Ion Trap

Collision-induced dissociation (CID) in linear ion traps is usually performed by applying a dipolar alternating current (AC) signal to one pair of electrodes, which results in ion excitation mainly in one direction. In this paper, we report simulation and experimental studies of the ion excitation in two coordinate directions by applying identical dipolar AC signals to two pairs of electrodes simultaneously. Theoretical analysis and simulation results demonstrate that the ion kinetic energy is higher than that using the conventional CID method. Experimental results show that more activation energy (as determined by the intensity ratio of the a4/b4 fragments from the CID of protonated leucine enkephalin) can be deposited into parent ions in this method. The dissociation rate constant in this method was about 3.8 times higher than that in the conventional method under the same experimental condition, at the Mathieu parameter qu (where u = x, y) value of 0.25. The ion fragmentation efficiency is also significantly improved. Compared with the conventional method, the smaller qu value can be used in this method to obtain the same internal energy deposited into ions. Consequently, the "low mass cut-off" is redeemed and more fragment ions can be detected. This excitation method can be implemented easily without changing any experimental parameters.

UV and IR spectroscopy of cold protonated leucine enkephalin

We have applied ultraviolet and infrared–ultraviolet (IR–UV) double resonance photofragment spectroscopy in a tandem mass spectrometer for the spectroscopic characterization of cryogenically-cooled protonated leucine enkephalin (H+-YGGFL), for the purposes of elucidating its three-dimensional structure. The primary UV-induced photofragmentation pathway following excitation of the tyrosine chromophore is loss of the tyrosine side chain (107Da). IR-enhanced photofragmentation via this channel makes IR–UV depletion spectroscopy difficult, and IR photofragment gain spectroscopy is used instead to record the infrared spectrum in the hydride stretch and amide I/II regions. By comparing the experimental spectrum with the predictions of DFT M05-2X/6-31+G(d) calculations, a single backbone structure was assigned that is similar to, but distinct from, that assigned in the recent work of Polfer et al. [15]. Additionally, the assigned structure’s theoretical cross-section is comparable to previous ion mobility results. The structure is characterized by a compact hydrogen-bonding architecture in which the peptide backbone self-solvates the N-terminal ammonium group carrying the charge. In addition to H-bonds to the tyrosine π cloud and the second glycine carbonyl oxygen, the ammonium group is involved in a series of cooperatively strengthened H-bonds between the N and C termini, linking the COOH group to the FL peptide bond. The resulting structure suggests some relevance to the fragmentation pathways of protonated YGGFL.

Femtosecond laser induced ionization and dissociation of gas-phase protonated leucine enkephalin

We have combined a tandem mass spectrometer with a 780nm fs-laser system to study photoionization and photofragmentation of trapped protonated leucine enkephalin cations for laser intensities between 2×1013W/cm2 and 1×1014W/cm2 and pulse durations of 15fs. In this intensity range, the transition from multiphoton ionization and excitation to tunneling ionization is expected to occur. The observed partial ion yield curves as a function of laser intensity exhibit a power-law dependence, indicating multiphoton absorption to be the dominating mechanism. Pump-probe studies were performed to investigate the time-evolution of the multiphoton ionization process. The partial ion yields of almost all fragmentation channels show a broad but distinct maximum at a delay-time of approximately 750fs. The particularly flat appearance of the pump-probe curves suggests that not a single resonance, but a broad distribution of resonances is involved.

How hot are your ions in TWAVE ion mobility spectrometry?

Effective temperatures of ions during traveling wave ion mobility spectrometry (TWIMS) analysis were measured using singly protonated leucine enkephalin dimer as a chemical thermometer by monitoring dissociation of the dimer into monomer, as well as the subsequent dissociation of monomer into a-, b-, and y-ions, as a function of instrumental parameters. At fixed helium cell and TWIMS cell gas flow rates, the extent of dissociation does not vary significantly with either the wave velocity or wave height, except at low (<500m/s) wave velocities that are not commonly used. Increasing the flow rate of nitrogen gas into the TWIMS cell and decreasing the flow rate of helium gas into the helium cell resulted in greater dissociation. However, the mobility distributions of the fragment ions formed by dissociation of the dimer upon injection into the TWIMS cell are nearly indistinguishable from those of fragment ions formed in the collision cell prior to TWIMS analysis for all TWIMS experiments. These results indicate that heating and dissociation occur when ions are injected into the TWIMS cell, and that the effective temperature subsequently decreases to a point at which no further dissociation is observed during the TWIMS analysis. An upper limit to the effective ion temperature of 449K during TWIMS analysis is obtained at a helium flow rate of 180mL/min, TWIMS flow rate of 80mL/min, and traveling wave height of 40V, which is well below previously reported values. Effects of ion heating in TWIMS on gas-phase protein conformation are presented.

Implementation of dipolar direct current (DDC) collision‐induced dissociation in storage and transmission modes on a quadrupole/time‐of‐flight tandem mass spectrometer

Means for effecting dipolar direct current collision-induced dissociation (DDC CID) on a quadrupole/time-of-flight in a mass spectrometer have been implemented for the broadband dissociation of a wide range of analyte ions. The DDC fragmentation method in electrodynamic storage and transmission devices provides a means for inducing fragmentation of ions over a large mass-to-charge range simultaneously. It can be effected within an ion storage step in a quadrupole collision cell that is operated as a linear ion trap or as ions are continuously transmitted through the collision cell. A DDC potential is applied across one pair of rods in the quadrupole collision cell of a QqTOF hybrid mass spectrometer to effect fragmentation. In this study, ions derived from a small drug molecule, a model peptide, a small protein, and an oligonucleotide were subjected to the DDC CID method in either an ion trapping or an ion transmission mode (or both). Several key experimental parameters that affect DDC CID results, such as time, voltage, low mass cutoff, and bath gas pressure, are illustrated with protonated leucine enkephalin. The DDC CID dissociation method gives a readily tunable, broadband tool for probing the primary structures of a wide range of analyte ions. The method provides an alternative to the narrow resonance conditions of conventional ion trap CID and it can access more extensive sequential fragmentation, depending upon conditions. The DDC CID approach constitutes a collision analog to infrared multiphoton dissociation (IRMPD).

Adaptation of a 3-D Quadrupole Ion Trap for Dipolar DC Collisional Activation

Means to allow for the application of a dipolar DC pulse to the end-cap electrodes of a three-dimensional (3-D) quadrupole ion trap for as short as a millisecond to as long as hundreds of milliseconds are described. The implementation of dipolar DC does not compromise the ability to apply AC waveforms to the end-cap electrodes at other times in the experiment. Dipolar DC provides a nonresonant means for ion acceleration by displacing ions from the center of the ion trap where they experience stronger rf electric fields, which increases the extent of micro-motion. The evolution of the product ion spectrum to higher generation products with time, as shown using protonated leucine enkephalin as a model protonated peptide, illustrates the broad-band nature of the activation. Dipolar DC activation is also shown to be effective as an ion heating approach in mimicking high amplitude short time excitation (HASTE)/pulsed Q dissociation (PQD) resonance excitation experiments that are intended to enhance the likelihood for observing low m/z products in ion trap tandem mass spectrometry.

Photodissociation of protonated leucine-enkephalin in the VUV range of 8–40 eV

Until now, photodissociation studies on free complex protonated peptides were limited to the UV wavelength range accessible by intense lasers. We have studied photodissociation of gas-phase protonated leucine-enkephalin cations for vacuum ultraviolet (VUV) photons energies ranging from 8 to 40 eV. We report time-of-flight mass spectra of the photofragments and various photofragment-yields as a function of photon energy. For sub-ionization energies our results are in line with existing studies on UV photodissociation of leucine-enkephalin. For photon energies exceeding 10 eV we could identify a new dissociation scheme in which photoabsorption leads to a fast loss of the tyrosine side chain. This loss process leads to the formation of a residual peptide that is remarkably cold internally.

Dissociation Kinetics of Singly Protonated Leucine Enkephalin Investigated by Time-Resolved Photodissociation Tandem Mass Spectrometry

The yields of post-source decay (PSD) and time-resolved photodissociation (PD) at 193 and 266 nm were measured for singly protonated leucine enkephalin ([YGGFL + H](+)), a benchmark in the study of peptide ion dissociation, by using tandem time-of-flight mass spectrometry. The peptide ion was generated by matrix-assisted laser desorption ionization (MALDI) using 2,5-dihydroxybenzoic acid as the matrix. The critical energy (E(0)) and entropy (DeltaS(++) at 1000 K) for the dissociation were determined by Rice-Ramsperger-Kassel-Marcus fit of the experimental data. MALDI was done for a mixture of YGGFL and Y(6) and the plume temperature determined by the kinetic analysis of [Y(6) + H](+) data were used to improve the precision of E(0) and DeltaS(++) for [YGGFL + H](+). E(0) and DeltaS(++) thus determined (E(0) = 0.67 +/- 0.08 eV, DeltaS(++) = -24.4 +/- 3.2 eu with 1 eu = 4.184 J K(-1)mol(-1)) were significantly different from those determined by blackbody infrared radiative dissociation (BIRD) (E(0) = 1.10 eV, DeltaS(++) = -14.9 eu), and by surface-induced dissociation (SID) (E(0) = 1.13 eV, DeltaS(double dagger) = -10.3 eu). Analysis of the present experimental data with the SID kinetics (and BIRD kinetics also) led to an unrealistic situation where not only PSD and PD but also MALDI-TOF signals could not be detected. As an explanation for the discrepancy, it was suggested that transition-state switching occurs from an energy bottleneck (SID/BIRD) to an entropy bottleneck (PSD/PD) as the internal energy increases.

Measuring Internal Energy Deposition in Collisional Activation Using Hydrated Ion Nanocalorimetry to Obtain Peptide Dissociation Energies and Entropies

The internal energy deposited in both on- and off-resonance collisional activation in Fourier transform ion cyclotron resonance mass spectrometry is measured with ion nanocalorimetry and is used to obtain information about the dissociation energy and entropy of a protonated peptide. Activation of Na(+)(H(2)O)(30) results in sequential loss of water molecules, and the internal energy of the activated ion can be obtained from the abundances of the product ions. Information about internal energy deposition in on-resonance collisional activation of protonated peptides is inferred from dissociation data obtained under identical conditions for hydrated ions that have similar m/z and degrees-of-freedom. From experimental internal energy deposition curves and Rice-Ramsperger-Kassel-Marcus (RRKM) theory, dissociation data as a function of collision energy for protonated leucine enkephalin, which has a comparable m/z and degrees-of-freedom as Na(+)(H(2)O)(30), are modeled. The threshold dissociation energies and entropies are correlated for data acquired at a single time point, resulting in a relatively wide range of threshold dissociation energies (1.1 to 1.7 eV) that can fit these data. However, this range of values could be significantly reduced by fitting data acquired at different dissociation times. By measuring the internal energy of an activated ion, the number of fitting parameters necessary to obtain information about the dissociation parameters by modeling these data is reduced and could result in improved accuracy for such methods.

Energetics and Dynamics of Fragmentation of Protonated Leucine Enkephalin from Time- and Energy-Resolved Surface-Induced Dissociation Studies

Dissociation of singly protonated leucine enkephalin (YGGFL) was studied using surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially configured for studying ion activation by collisions with surfaces. The energetics and dynamics of seven primary dissociation channels were deduced from modeling the time- and energy-resolved fragmentation efficiency curves for different fragment ions using an RRKM-based approach developed in our laboratory. The following threshold energies and activation entropies were determined in this study: E(0) = 1.20 eV and DeltaS++ = -20 eu(1) (MH(+)-->b(5)); E(0) = 1.14 eV and DeltaS++ = -14.7 eu (MH(+)-->b(4)); E(0) = 1.42 eV and DeltaS++ = -2.5 eu (MH(+)-->b(3)); E(0) = 1.30 eV and DeltaS++ = -4.1 eu (MH(+)-->a(4)); E(0) = 1.37 eV and DeltaS++ = -5.2 eu (MH(+)-->y ions); E(0) = 1.50 eV and DeltaS++ = 1.6 eu (MH(+)-->internal fragments); E(0) = 1.62 eV and DeltaS++ = 5.2 eu (MH(+)-->F). Comparison with Arrhenius activation energies reported in the literature demonstrated for the first time the reversal of the order of activation energies as compared to threshold energies for dissociation.

Internal energy distribution in electrospray ionization

Internal energies and energy distributions were studied using the 'survival yield' method developed previously. In addition to conventional benzylpyridinium salts, protonated esters (fragmenting by rearrangement) and protonated leucine enkephalin were also used, extending the validity of the technique. Fragmentation processes were studied in the cone voltage region and modeled by the RRKM-based MassKinetics program. The results show that the shapes of the energy distributions are similar to thermal distributions. The mean internal energies are very similar for all compound classes studied, and show a linear increase with collision energy in the 10-50 eV region.

Design and performance of a new FT-ICR cell operating at a temperature range of 77–438 K

A new ion cell for Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), which can be operated within the temperature range 77–438 K, has been designed and constructed. It has an elongated open-ended cylindrical configuration with capacitively coupled trapping electrodes. Fast and accurate thermal control of the cell is realized by embedding a heating element and a cooling pipe into the ceramic jacket holding the cell electrode plates. To determine the geometry factor, β, of the cell a novel empirical methodology has been developed that is applicable to any ICR cell. This was achieved by comparing breakdown diagrams of protonated leucine enkephalin obtained using the new cell with those obtained using a well characterized cell. Energy-resolved collision-activated dissociation (CAD) of protonated leucine enkephalin, performed using the new cell, was applied to probe the internal energy content of ions at different ICR cell temperatures. These experiments demonstrate that the trapped ion population reaches the preset temperature of the cell through thermal equilibration with the cell walls by blackbody infrared radiation. This has permitted FT-ICR-MS studies (dissociation or ion–molecule reactions, etc.) to be performed at a wide temperature range, including low temperatures.

Manipulating internal energy of protonated biomolecules in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The internal energy of protonated leucine enkephalin has been manipulated in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry with two newly designed pump-probe experiments. Blackbody infrared radiation was applied to pump an ion population into a well-defined internal energy distribution below the dissociation threshold. Following this pumping stage, the internal energy distribution was probed using on-resonance collisional activation to dissociate the ions. These pump-probe experiments were carried out in two different ways: (a) using on-resonance collisional activation with variable kinetic energies to dissociate the ions at a constant initial ion temperature (determining the precursor ion survival percentage as a function of kinetic energy) and (b) using on-resonance collisional activation with a constant kinetic energy to dissociate the ions at variable initial ion temperatures (to investigate the ion survival yield-initial ion temperature dependence). Using this approach, a detailed study of the effects of the initial ion temperature, the probing kinetic energy and the internal energy loss rate on the effective conversion efficiency of (laboratory-frame) kinetic energy to internal energy was conducted. This conversion efficiency was found to be dependent on the initial ion temperature. Depending on the experimental conditions the conversion efficiency (for collisions with argon) was estimated to be about 4.0 +/- 1.7%, which agrees with that obtained from a theoretical modeling. Finally, the reconstructed curves of the ion survival yield versus the mode of the (final) total internal energy distribution of the activated ion population (after pump and probe events) at different pump-probe conditions reveal the internal energy content of the activated ions.

Implementation of low-energy surface-induced dissociation (eV SID) and high-energy collision-induced dissociation (keV CID) in a linear sector-TOF hybrid tandem mass spectrometer

In the work reported here, an in-line sector-TOF (time-of-flight) tandem mass spectrometer with a coaxial detector was developed. An advantage of the present design is that the instrument implements both high-energy collision-induced dissociation (keV CID) and low-energy surface-induced dissociation (eV SID) with no hardware manipulations required to switch between the two activation methods. Surface characterization by secondary-ion mass spectrometry is also possible with this design. Gas phase collision-induced dissociation in this sector-TOF requires ion gating of the continuous ion beam and an ion buncher to bring the ions into spatial focus at the focal-point “source” of the offset parabolic field reflectron. The offset parabolic field reflectron provides good resolution over virtually the entire product ion mass range, with no need to step the reflector voltages. For surface-induced dissociation experiments, accomplished with a surface placed after the last reflectron electrode, the electrodes that comprise the CID reflectron are used for deceleration and acceleration of the ion beam before and after it collides with the surface, respectively. The results obtained show successful implementation of keV CID and eV SID in a JEOL HX110A/TOF mass spectrometer. Product ion mass accuracy is better than ±0.4 u for keV CID and ±0.1 u for eV SID. Typical resolutions achieved are 8000–12,000 (for selected ions), 1000 (for keV CID fragments), and 300–400 (for eV SID fragments). This difference in the CID and SID resolution is partially the result of the different total flight times in CID and SID experiments. On the basis of a direct comparison of keV CID and eV SID of protonated leucine enkephalin, SID experiments have a lower detection limit than CID experiments by at least an order of magnitude.

Bath gas temperature and the appearance of ion trap tandem mass spectra of high-mass ions

Bath gas temperature effects upon the appearance of ion trap tandem mass spectra of protonated leucine enkephalin have been studied under a variety of ion activation conditions and over a bath gas temperature range of 298–486 K. Bath gas temperature was found to have two possible effects upon the identities and abundances of product ions observed in tandem mass spectra. At high parent ion dissociation rates (>10 s −1) and short activation times (<30 ms), bath gas temperature principally affects the appearance of tandem mass spectra via its role in determining the kinetics for first generation product ion cooling. In the case of protonated leucine enkephalin, this is primarily reflected in the extent to which the first generation b 4 + ion fragments further to yield the a 4 + ion, the energy supplied for this process arising from the parent ion internal energy. Bath gas temperature is most likely to be an important factor in the appearance of tandem mass spectra collected under the conditions just mentioned, when a first generation product ion tends to fragment at rates comparable to or greater than the parent ion dissociation rate. At low parent ion dissociation rates (>10 s −1), relatively long ion activation times (<100 ms), and elevated bath gas temperatures, dissociation resulting from thermal activation of the first and higher generation fragments can make significant contributions to the tandem mass spectra.

Effective ion internal temperatures achieved via boundary activation in the quadrupole ion trap: protonated leucine enkephalin

Effective ion internal temperatures achieved via boundary activation in the quadrupole ion trap: protonated leucine enkephalin

Effective ion internal temperatures achieved via boundary activation in the quadrupole ion trap: protonated leucine enkephalin

The dissociation rate of protonated leucine enkephalin in a quadrupole ion trap was measured as a function of the proximity to which the ions are brought to a stability boundary. A bath gas temperature of 480 K was used so that a dissociation rate could be readily measured even when the parent ions were remote from a stability boundary. Changes in the dissociation rate as a function of d.c. potential applied to the ring electrode of the ion trap, used to bring ions close to a stability boundary, could then be attributed to the ‘boundary activation’ process. A relationship between dissociation rate and parent ion effective internal temperature, derived from a study involving conventional ion trap resonance excitation, was used to estimate effective ion internal temperatures achieved under the boundary activation conditions used here. Effective ion internal temperatures 170 K above the bath gas temperature could readily be achieved using boundary activation. However, the efficiency of the overall boundary activation experiment was compromised by an initial rapid parent ion loss that occurred upon application of the d.c. potential. After correction for this initial ion loss, dissociation rates and relatively low ion ejection rates could be measured. Effective ion internal temperatures achieved with boundary activation are similar to those observed previously using conventional resonance excitation. However, the generally poorer efficiencies associated with boundary activation that are observed with resonance excitation at high dissociation rates suggests that higher ion internal temperatures can be achieved with conventional resonance excitation. Copyright © 1999 John Wiley & Sons, Ltd.