Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes.

An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I2MS) method for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.

Image charge detection mass spectrometry: pushing the envelope with sensitivity and accuracy.

A novel image charge detection mass spectrometer (CDMS) with improved sensitivity and mass accuracy is described. The improved detector design and method of data analysis allow us to measure a reliable mass for a single macroion that is an order of magnitude smaller than previously achieved with CDMS. The apparatus employs an image charge detector array consisting of 22 detectors. The detectors are divided into two groups that can be floated at different potentials. The signals from the detector array are analyzed using a correlation approach to yield the velocities in the two groups of detectors and the charge. These quantities, together with the voltage difference between the two groups of detectors, provide a value for the mass. The mass, m/z, and charge distributions recorded for 300 kDa poly(ethylene oxide) (PEG) are presented. The mass distribution shows a peak at around 300 kDa with a width close to that expected from the polymer size distribution. In addition, there are broad peaks in the mass distribution at around 100 and 500 MDa. The 300 kDa ions have m/z ratios of ∼2 kDa/e, and the 100 and 500 MDa ions have m/z ratios of ∼40 kDa/e. The 100 and 500 MDa ions probably result from PEG aggregates that are either present in solution or the residue of large electrospray droplets.

Charge separation from the bursting of bubbles on water.

Film droplets formed from the bursting of 2.4 mm diameter bubbles on the surface of pure water are predominantly negatively charged. The charge generated per bubble varies chaotically; a few bubbles generate more than -3 × 10(6) elementary charges (e) but the vast majority generate much less. The average is -5 × 10(4)e/bubble, and it is not significantly affected by bubbling rate or temperature. The charge diminishes with increasing salt concentration and vanishes for concentrations above 10(-3) M. We propose a mechanism consistent with the observed charge separation. The model relies on the assumption that the surface of pure water has a slight excess of hydroxide ions. The charge separation results when water with entrained counterions (H(3)O(+)) flows out of the thinning film of the bubble cap, leaving behind the excess OH(-) on the surface. Addition of salt reduces the Debye length, and the charge separation mechanism becomes less effective as the Debye length becomes small compared with the film thickness. The excess charge near the surface of pure water is very small, around -4 nC/m(2).

STORI Plots Enable Accurate Tracking of Individual Ion Signals.

Charge detection mass spectrometry (CDMS) of low-level signals is currently limited to the analysis of individual ions that generate a persistent signal during the entire observation period. Ions that disintegrate during the observation period produce reduced frequency domain signal amplitudes, which lead to an underestimation of the ion charge state, and thus the ion mass. The charge assignment can only be corrected through an accurate determination of the time of ion disintegration. The traditional mechanisms for temporal signal analysis have severe limitations for temporal resolution, spectral resolution, and signal-to-noise ratios. Selective Temporal Overview of Resonant Ions (STORI) plots provide a new framework to accurately analyze low-level time domain signals of individual ions. STORI plots allow for complete correction of intermittent signals, the differentiation of single and multiple ions at the same frequency, and the association of signals that spontaneously change frequency.

Charge separation in the aerodynamic breakup of micrometer-sized water droplets.

Charged water droplets generated by electrospray, sonic spray, and a vibrating orifice aerosol generator (VOAG) have been studied by digital macrophotography and image charge detection mass spectrometry. Image charge detection mass spectrometry provides information on the droplet size, charge, and velocity after transmission through a capillary interface. The digital images provide the droplet size distribution before they enter the capillary. Droplets with 10-100 microm radii generated by sonic spray and VOAG are reduced to 2-3 microm radii by transmission through the capillary interface. The droplets from sonic spray and VOAG are much more highly charged than expected for random charging, and positive droplets are much more prevalent than negative. For positive mode electrospray, >99% of the detected droplets carry a positive charge, whereas for negative mode electrospray, <30% of the detected droplets carry a negative charge (i.e., >70% carry a positive charge). These observation can all be accounted for by the aerodynamic breakup of the droplets in the capillary interface. This breakup reduces the droplets to a terminal size at which point further breakup does not occur. Charge separation during droplet breakup is responsible for the relatively high charges on the sonic spray and VOAG droplets and for the preference for positively charged droplets. The charge separation can be explained using the bag mechanism for droplet breakup and the electrical bilayer at the surface of water.

Pulsed acceleration charge detection mass spectrometry: application to weighing electrosprayed droplets.

We describe a new approach to measuring the masses of individual macroions. The method employs a pulsed acceleration tube located between two sensitive image charge detectors. The charge and velocity of the macroion are recorded with the first image charge detector. The ion is pulse accelerated through a known voltage drop, and then the charge and velocity are remeasured using the second image charge detector. The mass of the ion is deduced from its charge and its initial and final velocities. The approach has been used to measure masses in the 10(10)-10(14) Da range with z = 10(3)-10(6) and m/z = 10(6)-10(9). It should be extendable to masses of <10(6) Da. We have used the method to determine the size and charge of water droplets transmitted through a capillary interface and an aperture interface. The droplets detected from the aperture interface are approximately 1 order of magnitude smaller in mass than those detected from the capillary interface. The droplets from both interfaces have relatively low charges, particularly with the capillary interface where they are only charged to a small fraction of the Rayleigh limit. These results suggest that the aerodynamic breakup of the droplets plays a significant role in the mechanism of electrospray ionization.

Negative droplets from positive electrospray.

Image charge detection has been used to measure the charge and velocity of individual electrosprayed water droplets. With a positive bias on the electrospray needle the majority of the droplets are, as expected, positively charged. However, a small fraction, surprisingly, carry a negative charge. Plausible explanations for the presence of the negatively charged droplets are discussed. In particular, we consider the possibility of the negatively charged droplets resulting from a bipolar fission process where the incorporation of a small negatively charged droplet between two larger positively charged progeny lowers the energy barrier for symmetric fission.

Reactivity of small Mo(x)Oy- clusters toward methane and ethane.

The reactions of Mo2Oy- suboxide clusters with both methane and ethane have been studied with a combination of mass spectrometry, anion photoelectron spectroscopy, and density functional theory calculations. Reactions were carried out under "gentle" and "violent" conditions. For methane, a number of products appeared under the gentler source conditions that were more logically attributed to dissociation of Mo2Oy- clusters upon reacting with methane to form MoCH2-, Mo(O)CH2-, and HMo(O2)CH3-. With ethane, products observed under the same gentle conditions were Mo(O)C2H2-, Mo(O)C2H4-, Mo(O2)C2H4-, and Mo(O2)(C2H5)2-. As expected, more products were observed when the reactions were carried out under violent conditions. The photoelectron spectra obtained for these species were compared to calculated adiabatic and vertical electron affinities and vibrational frequencies, leading to definitive structural assignments for several of the products.

Structures of Mo2Oy- and Mo2Oy (y=2, 3, and 4) studied by anion photoelectron spectroscopy and density functional theory calculations.

The competitive structural isomers of the Mo(2)O(y) (-)Mo(2)O(y) (y=2, 3, and 4) clusters are investigated using a combination of anion photoelectron (PE) spectroscopy and density functional theory calculations. The PE spectrum and calculations for MoO(3) (-)MoO(3) are also presented to show the level of agreement to be expected between the spectra and calculations. For MoO(3) (-) and MoO(3), the calculations predict symmetric C(3v) structures, an adiabatic electron affinity of 3.34 eV, which is above the observed value 3.17(2) eV. However, there is good agreement between observed and calculated vibrational frequencies and band profiles. The PE spectra of Mo(2)O(2) (-) and Mo(2)O(3) (-) are broad and congested, with partially resolved vibrational structure on the lowest energy bands observed in the spectra. The electron affinities (EA(a)s) of the corresponding clusters are 2.24(2) and 2.33(7) eV, respectively. Based on the calculations, the most stable structure of Mo(2)O(2) (-) is Y shaped, with the two Mo atoms directly bonded. Assignment of the Mo(2)O(3) (-) spectrum is less definitive, but a O-Mo-O-Mo-O structure is more consistent with overall electronic structure observed in the spectrum. The PE spectrum of Mo(2)O(4) (-) shows cleanly resolved vibrational structure and electronic bands, and the EA of the corresponding Mo(2)O(4) is determined to be 2.13(4) eV. The structure most consistent with the observed spectrum has two oxygen bridge bonds between the Mo atoms.