Multiplexed Charge Detection Mass Spectrometry for High-Throughput Single Ion Analysis of Large Molecules.

Applications of charge detection mass spectrometry (CDMS) for measuring the masses of large molecules, macromolecular complexes, and synthetic polymers that are too large or heterogeneous for conventional mass spectrometry measurements are made possible by weighing individual ions in order to avoid interferences between ions. Here, a new multiplexing method that makes it possible to measure the masses of many ions simultaneously in CDMS is demonstrated. Ions with a broad range of kinetic energies are trapped. The energy of each ion is obtained from the ratio of the intensity of the fundamental to the second harmonic frequencies of the periodic trapping motion making it possible to measure both the m/ z and charge of each ion. Because ions with the exact same m/ z but with different energies appear at different frequencies, the probability of ion-ion interference is significantly reduced. We show that the measured mass of a protein complex consisting of 16 protomers, RuBisCO (517 kDa), is not affected by the number of trapped ions with up to 21 ions trapped simultaneously in these experiments. Ion-ion interactions do not affect the ion trapping lifetime up to 1 s, and there is no influence of the number of ions on the measured charge-state distribution of bovine serum albumin (66.5 kDa), indicating that ion-ion interactions do not adversely affect any of these measurements. Over an order of magnitude gain in measurement speed over single ion analysis is demonstrated, and significant additional gains are expected with this multi-ion measurement method.

Simultaneous Measurements of Mass and Collisional Cross-Section of Single Ions with Charge Detection Mass Spectrometry.

The masses and mobilities of single multiply charged ions of cytochrome c, ubiquitin, myoglobin, and bovine serum albumin formed by electrospray ionization are measured using charge detection mass spectrometry (CDMS). Single ions are trapped and repeatedly measured as they oscillate inside an electrostatic ion trap with cone electrodes for up to the maximum trapping time set at 500 ms. The histograms of the many single ion oscillation frequencies have resolved peaks that correspond to the different charge states of each protein. The m/z of each ion is determined from the initial oscillation frequency histogram, and the evolution of the ion energy with time is obtained from the changing frequency. A short-time Fourier transform of the time-domain data indicates that the increase in ion frequency occurs gradually with time with occasional sudden jumps in frequency. The frequency jumps are similar for each protein and may be caused by collision-induced changes in the ion trajectory. The rate of the gradual frequency shift increases with protein mass and charge state. This gradual frequency change is due to ion energy loss from collisions with the background gas. The total energy lost by an ion is determined from the latter frequency shifts normalized to a 500 ms lifetime, and these values increase nearly linearly with measured collisional cross-sections for these protein ions. These results show that the mass and collisional cross-section of single multiply charged ions can be obtained from these CDMS measurements by using proteins with known collisional cross-sections for calibration.

Mass, mobility and MSn measurements of single ions using charge detection mass spectrometry.

Charge detection mass spectrometry is used to measure the mass, charge, MSn and mobility of an individual ion produced by electrospray ionization of a 8 MDa polyethylene glycol sample. The charge detection mass spectrometer is an electrostatic ion trap that uses cone electrodes and a single tube detector and can detect ions for up to the full trapping time of 4.0 s. The time-domain signal induced on the detector tube by a single multiply charged ion can be complex owing to sequential fragmentation of the original precursor ion as well as increasing oscillation frequencies of the single ion owing to collisions with background gas that reduce the kinetic energy of the ion inside the trap. Simulations show that the ratio of the time for the ion to turn around inside the cone region of the trap to the time for the ion to travel through the detector tube is constant with m/z and increases with the ion energy per charge. By measuring this ratio, the kinetic energy of an ion can be obtained with good precision (∼1%) and this method to measure ion kinetic energies eliminates the necessity of ion energy selection prior to trapping for high precision mass measurement of large molecules in complex mixtures. This method also makes it possible to measure the masses of each sequential fragment ion formed from the original precursor ion. MS7 of a single multiply charged PEG molecule is demonstrated, and from these ion energy measurements and effects of collisions on the ion motion inside the trap, information about the ion mobility of the precursor ion and its fragments is obtained.

Single Particle Analyzer of Mass: A Charge Detection Mass Spectrometer with a Multi-Detector Electrostatic Ion Trap.

A new charge detection mass spectrometer that combines array detection and electrostatic ion trapping to repeatedly measure the masses of single ions is described. This instrument has four detector tubes inside an electrostatic ion trap with conical electrodes (cone trap) to provide multiple measurements of an ion on each pass through the trap resulting in a signal gain over a conventional trap with a single detection tube. Simulations of a cone trap and a dual ion mirror trap design indicate that more passes through the trap per unit time are possible with the latter. However, the cone trap has the advantages that ions entering up to 2 mm off the central axis of the trap are still trapped, the trapping time is less sensitive to the background pressure, and only a narrow range of energies are trapped so it can be used for energy selection. The capability of this instrument to obtain information about the molecular weight distributions of heterogeneous high molecular weight samples is demonstrated with 8 MDa polyethylene glycol (PEG) and 50 and 100 nm amine modified polystyrene nanoparticle samples. The measured mass distribution of the PEG sample is centered at 8 MDa. The size distribution obtained from mass measurements of the 100 nm nanoparticle sample is similar to the size distribution obtained from transmission electron microscopy (TEM) images, but most of the smaller nanoparticles observed in TEM images of the 50 nm nanoparticles do not reach a sufficiently high charge to trigger the trap on a single pass and be detected by the mass spectrometer. With the maximum trapping time set to 100 ms, the charge uncertainty is as low as ±2 charges and the mass uncertainty is approximately 2% for PEG and polystyrene ions.

Characterization of an iron-ruthenium interaction in a ferrocene diamide complex.

Reaction of [fc(NH2)2]RuCl2(PPh3)2 (fc = 1,1'-ferrocenylene) with 2 equiv of KO(t)Bu led to the formation of a diamido ruthenium complex, [fc(NH)2]Ru(PPh3)2, whose solid-state molecular structure revealed a short Fe-Ru distance. A metal-to-metal charge transfer band was observed in the electronic absorption spectrum of [fc(NH)2]Ru(PPh3)2. The Fe-Ru interaction was characterized by resonance Raman spectroscopy for the first time and also by (1)H NMR, UV-vis, NIR, Mössbauer spectroscopy, and X-ray crystallography. Density functional theory (DFT) calculations including natural bond order analysis, Bader's atom in molecules method, and time-dependent DFT (TDDFT) provided further support that the iron-ruthenium bond is a weak donor-acceptor interaction with iron acting as the Lewis base.

Effects of Individual Ion Energies on Charge Measurements in Fourier Transform Charge Detection Mass Spectrometry (FT-CDMS).

A method to correct for the effect of ion energy on charge measurements of individual ions trapped and weighed with charge detection mass spectrometry (CDMS) is demonstrated. Ions with different energies induce different signal patterns inside an electrostatic ion trap. The sum of the amplitudes of the fundamental and second harmonic frequencies in the Fourier transform of the induced signal, which has been used to obtain the ion charge, depends on both ion energy and charge. The amplitudes of the fundamental frequencies of ions increase over time as ions lose energy by collisions with background gas and solvent loss from larger ions. Model ion signals are simulated with the same time-domain amplitude at different energies and frequencies and the resulting fundamental frequency amplitudes are used to normalize real ion signals for energy and frequency effects. The fundamental frequency amplitude decreases dramatically below 20 kHz and increases by ~ 17% from the highest energy to lowest energy that is stable with a given trap potential at all frequencies. Normalizing the fundamental frequency amplitude with the modeled amplitudes removes the systematic changes in the charge measurement of polyethylene glycol (PEG) and other ions and makes it possible to signal average the amplitude over long times, which reduces the charge uncertainty to 0.04% for a PEG ion for a 500-ms measurement. This method improves charge measurement accuracy and uncertainty, which are important for high-accuracy mass measurement with CDMS. Graphical abstract ᅟ.

Determining Energies and Cross Sections of Individual Ions Using Higher-Order Harmonics in Fourier Transform Charge Detection Mass Spectrometry (FT-CDMS).

A general method for in situ measurements of the energy of individual ions trapped and weighed using charge detection mass spectrometry (CDMS) is described. Highly charged (> 300 e), individual polyethylene glycol (PEG) ions are trapped and oscillate within an electrostatic trap, producing a time domain signal. A segmented Fourier transform (FT) of this signal yields the temporal evolution of the fundamental and harmonic frequencies of ion motion throughout the 500-ms trap time. The ratio of the fundamental frequency and second harmonic (HAR) depends on the ion energy, which is an essential parameter for measuring ion mass in CDMS. This relationship is calibrated using simulated ion signals, and the calibration is compared to the HAR values measured for PEG ion signals where the ion energy was also determined using an independent method that requires that the ions be highly charged (> 300 e). The mean error of 0.6% between the two measurements indicates that the HAR method is an accurate means of ion energy determination that does not depend on ion size or charge. The HAR is determined dynamically over the entire trapping period, making it possible to observe the change in ion energy that takes place as solvent evaporates from the ion and collisions with background gas occur. This method makes it possible to measure mass changes, either from solvent evaporation or from molecular fragmentation (MSn), as well as the cross sections of ions measured using CDMS. Graphical Abstract.

Transfer hydrogenation with a ferrocene diamide ruthenium complex.

The use of a 1,1'-ferrocenediamide ruthenium complex as a mediator for base-free transfer hydrogenation is reported. Ketones were transformed to their respective alcohols at room temperature in 36-99% conversions with turnover frequencies up to 339 h(-1).