Experimental Investigation of the 2D Ion Beam Profile Generated by an ESI Octopole-QMS System

In this paper, we have employed an ion imaging approach to investigate the behavior of ions exiting from a quadrupole mass spectrometer (QMS) system that employs a radio frequency octopole ion guide before the QMS. An in-vacuum active pixel detector (Timepix) is employed at the exit of the QMS to image the ion patterns. The detector assembly simultaneously records the ion impact position and number of ions per pixel in every measurement frame. The transmission characteristics of the ion beam exiting the QMS are studied using this imaging detector under different operating conditions. Experimental results confirm that the ion spatial distribution exiting the QMS is heavily influenced by ion injection conditions. Furthermore, ion images from Timepix measurements of protein standards demonstrate the capability to enhance the quality of the mass spectral information and provide a detailed insight in the spatial distribution of different charge states (and hence different m/z) ions exiting the QMS.      

Carbonation and Other Super Saturated Gases as Solution Modifiers for Improved Sensitivity in Solvent Assisted Ionization Inlet (SAII) and ESI

Solvent Assisted Ionization Inlet (SAII) produces ions in a heated inlet to a mass spectrometer from aqueous and aqueous/organic solutions with high sensitivity. However, the use of acid modifiers, which typically aids electrospray ionization, generally results in ion suppression in SAII. Here we demonstrate that the use of carbonation and other super-saturated gases as solution modifiers increases analyte ion abundance and reduces metal cation adduction in SAII. Carbonation is also found to enhance electrospray ionization. The mechanistic and practical utility of carbonation in mass spectrometry is addressed.      

Dual-Channel Electrospray Microchip

A dual-channel electrospray microchip has been developed for electrospray ionization mass spectrometry (ESI-MS) where aqueous samples are mixed at the Taylor cone with an organic buffer. Due to the fast and effective mixing in the Taylor cone, the aqueous sample can be well ionized with a high ion intensity. The influence of geometric parameters such as the distance between the two microchannels at their junction at the tip of the emitter has been investigated together with chemical parameters such as the organic buffer composition.      

Gas-Phase Intramolecular Protein Crosslinking via Ion/Ion Reactions: Ubiquitin and a Homobifunctional sulfo-NHS Ester

Gas-phase intra-molecular crosslinking of protein ubiquitin cations has been demonstrated via ion/ion reactions with anions of a homobifunctional N-hydroxysulfosuccinimide (sulfo-NHS) ester reagent. The ion/ion reaction between multiply-protonated ubiquitin and crosslinker monoanions produces a stable, charge-reduced complex. Covalent crosslinking is indicated by the consecutive loss of 2 molecules of sulfo-NHS under ion trap collisional activation conditions. Covalent modification is verified by the presence of covalently crosslinked sequence ions produced by ion-trap collision-induced dissociation of the ion generated from the losses of sulfo-NHS. Analysis of the crosslinked sequence fragments allows for the localization of crosslinked primary amines, enabling proximity mapping of the gas-phase 3-D structures. The presence of two unprotonated reactive sites within the distance constraint of the crosslinker is required for successful crosslinking. The ability to covalently crosslink is, therefore, sensitive to protein charge state. As the charge state increases, fewer reactive sites are available and protein structure is more likely to become extended because of intramolecular electrostatic repulsion. At high charge states, the reagent shows little evidence for covalent crosslinking but does show evidence for ‘electrostatic crosslinking’ in that the binding of the sulfonate groups to the protein is sufficiently strong that backbone cleavages are favored over reagent detachment under ion trap collisional activation conditions.      

Traveling Wave Ion Mobility Mass Spectrometry Study of Low Generation Polyamidoamine Dendrimers

We reported the use of ion mobility (IM) combined with mass spectrometry (MS) as an analytical tool to investigate low generation polyamidoanine (PAMAM) dendrimers. This analytical approach has been employed to separate ions of defective structures with different charge state but exactly the same m/z value. Tandem mass spectrometry (MS/MS) after IM separation allowed a comprehensive structural characterization of defective dendrimers. In addition, IM was used to evaluate the collision cross-sections of ions of perfect dendrimers. They showed a good correlation with calculated collision cross-sections obtained by the trajectory method (TM) and were also consistent with dimensions reported by other established analytical methods.      

A Simple Analytical Model for Predicting the Detectable Ion Current in Ion Mobility Spectrometry Using Corona Discharge Ionization Sources

Corona discharge ionization sources are often used in ion mobility spectrometers (IMS) when a non-radioactive ion source with high ion currents is required. Typically, the corona discharge is followed by a reaction region where analyte ions are formed from the reactant ions. In this work, we present a simple yet sufficiently accurate model for predicting the ion current available at the end of this reaction region when operating at reduced pressure as in High Kinetic Energy Ion Mobility Spectrometers (HiKE-IMS) or most IMS-MS instruments. It yields excellent qualitative agreement with measurement results and is even able to calculate the ion current within an error of 15%. Additional interesting findings of this model are the ion current at the end of the reaction region being independent from the ion current generated by the corona discharge and the ion current in High Kinetic Energy Ion Mobility Spectrometers (HiKE-IMS) growing quadratically when scaling down the length of the reaction region.

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Characterization of Electron Transfer Dissociation in the Orbitrap Velos HCD Cell

Electron transfer dissociation (ETD) is commonly employed in ion traps utilizing rf fields that facilitate efficient electron transfer reactions. Here, we explore performing ETD in the HCD collision cell on an Orbitrap Velos instrument by applying a static DC gradient axially to the rods. This gradient enables simultaneous three dimensional, charge sign independent, trapping of cations and anions, initiating electron transfer reactions in the center of the HCD cell where oppositely charged ions clouds overlap. Here, we evaluate this mode of operation for a number of tryptic peptide populations and the top-down sequence analysis of ubiquitin. Our preliminary data show that performing ETD in the HCD cell provides similar fragmentation as ion trap-ETD but requires further optimization to match performance of ion trap-ETD.      

Fischer Indole Synthesis in the Gas Phase,the Solution Phase,and at the Electrospray Droplet Interface

Previous reports have shown that reactions occurring in the microdroplets formed during electrospray ionization can, under the right conditions, exhibit significantly greater rates than the corresponding bulk solution-phase reactions. The observed acceleration under electrospray ionization could result from a solution-phase, a gas-phase, or an interfacial reaction. This study shows that a gas-phase ion/molecule (or ion/ion) reaction is not responsible for the observed rate enhancement in the particular case of the Fischer indole synthesis. The results show that the accelerated reaction proceeds in the microdroplets, and evidence is provided that an interfacial process is involved.

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Matrix Assisted Ionization in Vacuum, a Sensitive and Widely Applicable Ionization Method for Mass Spectrometry

An astonishingly simple new method to produce gas-phase ions of small molecules as well as proteins from the solid state under cold vacuum conditions is described. This matrix assisted ionization vacuum (MAIV) mass spectrometry (MS) method produces multiply charged ions similar to those that typify electrospray ionization (ESI) and uses sample preparation methods that are nearly identical to matrix-assisted laser desorption/ionization (MALDI). Unlike these established methods, MAIV does not require a laser or voltage for ionization, and unlike the recently introduced matrix assisted ionization inlet method, does not require added heat. MAIV-MS requires only introduction of a crystalline mixture of the analyte incorporated with a suitable small molecule matrix compound such as 3-nitrobenzonitrile directly to the vacuum of the mass spectrometer. Vacuum intermediate pressure MALDI sources and modified ESI sources successfully produce ions for analysis by MS with this method. As in ESI-MS, ion formation is continuous and, without a laser, little chemical background is observed. MAIV, operating from a surface offers the possibility of significantly improved sensitivity relative to atmospheric pressure ionization because ions are produced in the vacuum region of the mass spectrometer eliminating losses associated with ion transfer from atmospheric pressure to vacuum. Mechanistic aspects and potential applications for this new ionization method are discussed.      

Ion Mobility Separation of Peptide Isotopomers

Differential or field asymmetric waveform ion mobility spectrometry (FAIMS) operating at high electric fields fully resolves isotopic isomers for a peptide with labeled residues. The naturally present isotopes, alone and together with targeted labels, also cause spectral shifts that approximately add for multiple heavy atoms. Separation qualitatively depends on the gas composition. These findings may enable novel strategies in proteomic and metabolomic analyses using stable isotope labeling.

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Laserspray and Matrix-Assisted Ionization Inlet Coupled to High-Field FT-ICR Mass Spectrometry for Peptide and Protein Analysis

We present the first coupling of laser spray ionization inlet (LSII) and matrix assisted ionization inlet (MAII) to high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) for generation of electrospray-like ions to take advantage of increased sensitivity, mass range, and mass resolving power afforded by multiple charging. We apply the technique to top-down protein analysis and characterization of metalloproteins. We also present a novel method for generation of multiply-charged copper–peptide complexes with varying degrees of copper adduction by LSII. We show an application of the generated copper–peptide complexes for protein charge state and molecular weight determination, particularly useful for an instrument such as a linear ion trap mass analyzer.      

Gas-Phase Fragmentation of Oligoproline Peptide Ions Lacking Easily Mobilizable Protons

The fragmentation of peptides containing quaternary ammonium group, but lacking easily mobilizable protons, was examined with the aid of deuterium-labeled analogs and quantum-chemical modeling. The fragmentation of oligoproline containing quaternary ammonium group involves the mobilization of hydrogens localized at α- and γ- or δ-carbon atoms in the pyrrolidine ring of proline. The study of the dissociation pattern highlights the unusual proline residue behavior during MS/MS experiments of peptides.      

Rapid Characterization of Microorganisms by Mass Spectrometry—What Can Be Learned and How?

Strategies for the rapid and reliable analysis of microorganisms have been sought to meet national needs in defense, homeland security, space exploration, food and water safety, and clinical diagnosis. Mass spectrometry has long been a candidate technique because it is extremely rapid and can provide highly specific information. It has excellent sensitivity. Molecular and fragment ion masses provide detailed fingerprints, which can also be interpreted. Mass spectrometry is also a broad band method—everything has a mass—and it is automatable. Mass spectrometry is a physiochemical method that is orthogonal and complementary to biochemical and morphological methods used to characterize microorganisms.      

Structural Analysis of Triacylglycerols by Using a MALDI-TOF/TOF System with Monoisotopic Precursor Selection

A new MALDI-TOF/TOF system with monoisotopic precursor selection was applied to the analysis of triacylglycerols in an olive oil sample. Monoisotopic precursor selection made it possible to obtain product-ion mass spectra without interference from species that differed by a single double bond. Complete structure determination of all triacylglycerols, including structural isomers, was made possible by interpreting the charge-remote fragmentation resulting from high-energy collision-induced dissociation (CID) of the sodiated triacylglycerols.      

Study of In-Trap Ion Clouds by Ion Trajectory Simulations

Gaussian distribution has been utilized to describe the global number density distribution of ion cloud in the Paul trap, which is known as the thermal equilibrium theory and widely used in theoretical modeling of ion clouds in the ion traps. Using ion trajectory simulations, however, the ion clouds can now also be treated as a dynamic ion flow field and the location-dependent features could now be characterized. This study was carried out to better understand the in-trap ion cloud properties, such as the local particle velocity and temperature. The local ion number densities were found to be heterogeneously distributed in terms of mean and distribution width; the velocity and temperature of the ion flow varied with pressure depending on the flow type of the neutral molecules; and the “quasi-static” equilibrium status can only be achieved after a certain number of collisions, for which the time period is pressure-dependent. This work provides new insights of the ion clouds that are globally stable but subjected to local rf heating and collisional cooling.

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Study of Nonlinear Resonance Effect in Paul Trap

In this article, we investigated the nonlinear resonance effect in the Paul trap with a superimposed hexapole field, which was assumed as a perturbation to the quadrupole field. On the basis of the Poincare-Lighthill-Kuo (PLK) perturbation method, ion motional equation, known as nonlinear Mathieu equation (NME) was expressed as the addition of approximation equations in terms of perturbation order. We discussed the frequency characteristics of ion axial-radial (z-r) coupled motion in the nonlinear field, derived the expressions of ion trajectories and nonlinear resonance conditions, and found that the mechanism of nonlinear resonance is similar to the normal resonance. The frequency spectrum of ion motion in nonlinear field includes not only the natural frequency series but also nonlinear introduced frequency series, which provide the driving force for the nonlinear resonance. The nonlinear field and the nonlinear effects are inevitable in practical ion trap experiments. Our method provides better understanding of these nonlinear effects and would be helpful for the instrumentation for ion trap mass spectrometers.      

Development of an Ultra-High Performance Multi-Turn TOF-SIMS/SNMS System “MULTUM-SIMS/SNMS”

A new system incorporating a multi-turn time-of-flight secondary ion/sputtered neutral mass spectrometer (TOF-SIMS/SNMS) with laser post-ionization was designed and constructed. This system consists of a gallium focused ion beam, femtosecond (fs) laser for post-ionization, and multi-turn TOF mass spectrometer. When laser post-ionization was used, the secondary ion signal strengths for several metals increased by up to 650 times, and were greater than the values obtained in conventional TOF-SIMS experiments. Use of the multi-turn mass spectrometer resulted in an increase in mass resolving power with increase in the total TOF. The mass resolving power reached to 23,000 after 800 multi-turn cycles, corresponding to a flight path length of 1040 m. These results indicated that this system is very effective for the analysis of valuable materials such as space samples with high sensitivity, high mass resolving power, and high lateral resolution.      

The Effect of Organic Modifiers on Electrospray Ionization Charge-State Distribution and Desorption Efficiency for Oligonucleotides

The chemical composition of the solution has a critical impact on the electrospray desorption efficiency of oligonucleotides. Several physiochemical properties of various organic modifiers were investigated with respect to their role in the desorption process of oligonucleotides. The Henry’s Law Constant, which reflects the volatility of alkylamines, was found to have a prominent effect on both the electrospray charge state distribution and desorption efficiency of oligonucleotides. Alkylamines with higher $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values such as hexylamine, piperidine, and imidazole reduced the charge state distribution by forming complexes with the oligonucleotide and dissociating from it in the gas phase, while alkylamines with extremely low $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values reduced the electrospray charge state distribution by facilitating ion emission at an earlier stage of the electrospray desorption process. Ion-pairing agents with moderate $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values do not alter the electrospray charge state distribution of oligonucleotides and their ability to enhance oligonucleotide ionization followed the order of decreasing $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values. The Henry’s Law Constant also correlated to the impact of the acidic modifiers on oligonucleotide ionization efficiency. Ionization enhancement effects were observed with hexafluoroisopropanol, and this effect was attributed to its low $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ and moderate acidity. The comprehensive effects of both alkylamine and hexafluoroisoproapnol on the electrospray ionization desorption of oligonucleotides were also evaluated, and acid-base equilibrium was found to play a critical role in determining these effects.      

Extracting Charge and Mass Information from Highly Congested Mass Spectra Using Fourier-Domain Harmonics

Native mass spectra of large, polydisperse biomolecules with repeated subunits, such as lipoprotein Nanodiscs, can often be challenging to analyze by conventional methods. The presence of tens of closely spaced, overlapping peaks in these mass spectra can make charge state, total mass, or subunit mass determinations difficult to measure by traditional methods. Recently, we introduced a Fourier Transform-based algorithm that can be used to deconvolve highly congested mass spectra for polydisperse ion populations with repeated subunits and facilitate identification of the charge states, subunit mass, charge-state-specific, and total mass distributions present in the ion population. Here, we extend this method by investigating the advantages of using overtone peaks in the Fourier spectrum, particularly for mass spectra with low signal-to-noise and poor resolution. This method is illustrated for lipoprotein Nanodisc mass spectra acquired on three common platforms, including the first reported native mass spectrum of empty “large” Nanodiscs assembled with MSP1E3D1 and over 300 noncovalently associated lipids. It is shown that overtone peaks contain nearly identical stoichiometry and charge state information to fundamental peaks but can be significantly better resolved, resulting in more reliable reconstruction of charge-state-specific mass spectra and peak width characterization. We further demonstrate how these parameters can be used to improve results from Bayesian spectral fitting algorithms, such as UniDec.

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para-Nitroaniline is a Promising Matrix for MALDI-MS Imaging on Intermediate Pressure MS Systems

para-Nitroaniline (PNA) is presented as a promising matrix for matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) on an intermediate-pressure ion source (~1 Torr) QqTOF instrument using an Nd:YVO₄ laser operated at 5 kHz. An imaging study was carried out to determine the utility of PNA at this pressure by analyzing 14 tissue sections. We demonstrate acquisition of high-quality imaging data over a 6-h period in the ion source. In this study, comparisons were made between PNA and α-cyano-4-hydroxycinnamic acid (CHCA) in positive ion mode to demonstrate the utility of PNA in these circumstances. PNA performed as well as or better than CHCA in terms of lipid ion intensities, resulting in lower levels of ion fragmentation and in lower incidences of analyte migration at the edges of the tissue sections when using airspray matrix deposition.