Nanospray Desorption Electrospray Ionization (Nano-DESI) Mass Spectrometry Imaging with High Ion Mobility Resolution.

Untargeted separation of isomeric and isobaric species in mass spectrometry imaging (MSI) is challenging. The combination of ion mobility spectrometry (IMS) with MSI has emerged as an effective strategy for differentiating isomeric and isobaric species, which substantially enhances the molecular coverage and specificity of MSI experiments. In this study, we have implemented nanospray desorption electrospray ionization (nano-DESI) MSI on a trapped ion mobility spectrometry (TIMS) mass spectrometer. A new nano-DESI source was constructed, and a specially designed inlet extension was fabricated to accommodate the new source. The nano-DESI-TIMS-MSI platform was evaluated by imaging mouse brain tissue sections. We achieved high ion mobility resolution by utilizing three narrow mobility scan windows that covered the majority of the lipid molecules. Notably, the mobility resolution reaching up to 300 in this study is much higher than the resolution obtained in our previous study using drift tube IMS. High-resolution TIMS successfully separated lipid isomers and isobars, revealing their distinct localizations in tissue samples. Our results further demonstrate the power of high-mobility-resolution IMS for unraveling the complexity of biomolecular mixtures analyzed in MSI experiments.

High-Throughput Mass Spectrometry Imaging with Dynamic Sparse Sampling.

Mass spectrometry imaging (MSI) enables label-free mapping of hundreds of molecules in biological samples with high sensitivity and unprecedented specificity. Conventional MSI experiments are relatively slow, limiting their utility for applications requiring rapid data acquisition, such as intraoperative tissue analysis or 3D imaging. Recent advances in MSI technology focus on improving the spatial resolution and molecular coverage, further increasing the acquisition time. Herein, a deep learning approach for dynamic sampling (DLADS) was employed to reduce the number of required measurements, thereby improving the throughput of MSI experiments in comparison with conventional methods. DLADS trains a deep learning model to dynamically predict molecularly informative tissue locations for active mass spectra sampling and reconstructs high-fidelity molecular images using only the sparsely sampled information. Experimental hardware and software integration of DLADS with nanospray desorption electrospray ionization (nano-DESI) MSI is reported for the first time, which demonstrates a 2.3-fold improvement in throughput for a linewise acquisition mode. Meanwhile, simulations indicate that a 5-10-fold throughput improvement may be achieved using the pointwise acquisition mode.

High-throughput screening of organic reactions in microdroplets using desorption electrospray ionization mass spectrometry (DESI-MS): hardware and software implementation.

This study describes an automated system used for high throughput screening of reaction conditions based on accelerated reactions occurring in small volumes of reagents. Reaction mixtures are prepared in array format using a fluid handling robot and spotted on a flat polytetrafluoroethylene plate at densities up to 6144 per plate. The reaction and analysis steps are performed simultaneously using desorption electrospray ionization (DESI) to release microdroplets containing the reaction mixture from the plate for reaction prior to arrival at a mass spectrometer. Analysis rates are up to 1 reaction mixture per second and data are recorded in real time using an ion trap mass spectrometer. Beacon compounds are used to triangulate position on the plate and this allows tandem mass spectrometry (MS/MS) to be performed on confirm products of interest. Custom software allows the user to control the system. It is also used to receive data from the DESI mass spectrometer to screen the spectra for compounds of interest, to perform MS/MS and to save data. This custom software also communicates with the software controlling the fluid handling robot (Biomek i7) as well as the Beckman software used to prepare reaction mixtures and also the software that controls the solvent used as the DESI spray. Data were recorded for N-alkylation, N-acylation and N-sulfonylation reactions in three 8 hour experiments on successive days to establish the ruggedness and repeatability of the system. Repeatability is high (94-97%) over this period with false negative 6% (depending on noise threshold chosen). Plates containing 384 reaction mixtures are analyzed in 7 min by moving the DESI sprayer in steps under the sprayer instead of continuously.

Integration of a Multichannel Pulsed-Valve Inlet System to a Linear Quadrupole Ion Trap Mass Spectrometer for the Rapid Consecutive Introduction of Nine Reagents for Diagnostic Ion/Molecule Reactions.

Gas-phase ion/molecule reactions have been used extensively for the structural elucidation of organic compounds in tandem mass spectrometry. Reagents for ion/molecule reactions can be introduced into a mass spectrometer via a continuous flow apparatus or through a pulsed inlet system. However, most of these approaches enable the use of only a single reagent at a time. In this work, a multichannel pulsed-valve inlet system was developed for the rapid consecutive introduction of up to nine different reagents or reagent systems into a linear quadrupole ion trap mass spectrometer for diagnostic gas-phase ion/molecule reactions. Automated triggering of the pulsed valves enabled these experiments to be performed on the high-performance liquid chromatography (HPLC) time scale. This enables high-throughput screening of several functionalities in analytes as they elute from an HPLC column.

Electroosmotically driven solution mixing in borosilicate theta glass nESI emitters.

The use of borosilicate theta glass capillaries as nanoelectrospray ionization emitters has recently been demonstrated as a method for mixing two solutions as they are sprayed into the mass spectrometer for analysis. All previous experiments resulted in a solution mixing timescale limited to the time the analytes spend in the Taylor cone and subsequent droplets (i.e. sub-millisecond timescale). In an effort to extend the solution mixing timescale to the milliseconds regime, we demonstrate that solution can be moved from one channel of the theta tip to the opposite channel via electroosmosis by applying a potential difference between the two wire electrodes inserted into each channel of the theta tip. First, we establish that electroosmosis is responsible for solution movement using fluorescence microscopy to track fluorescent tracer dyes. We then demonstrate the utility of this technique in varying the extent of denaturation of holomyoglobin to apomyoglobin on the millisecond timescale just prior to analysis by mass spectrometry. Finally, we induce additional turbulence for better mixing by applying a square wave potential to one of the wire electrodes while holding the opposite wire at a constant voltage between the low and high potentials of the square wave. This experiment was found to provide nearly complete mixing after a single cycle of the square wave. The use of electroosmosis significantly expands the flexibility of theta tips for altering solutions prior to nESI without the need for off-line sample manipulation. Copyright © 2015 John Wiley & Sons, Ltd.

Tandem mass spectrometry in an electrostatic linear ion trap modified for surface-induced dissociation.

A variety of ion traps are used in mass spectrometry. A key feature shared by most of them is the ability to perform tandem mass spectrometry (MS/MS). The Orbitrap is perhaps the most notable ion trap in which MS/MS has yet to be performed. An electrostatic linear ion trap (ELIT) is analogous to an orbitrap in that ions are trapped using solely electrostatic fields. However, the relatively simple ion motion within an ELIT facilitates analysis of fragment ions produced within the device. In this report, we describe an ELIT to which we have added a target for surface induced dissociation (SID). When combined with our previously described method for isolating a precursor ion trapped in an ELIT,1 this apparatus enables MS/MS to be performed. Measurement of product ion m/z is facilitated by the fact that the ELIT is isochronous over the energy range of 1850-2000 eV so that changes to ion energy during SID do not cause major m/z shifts. We demonstrate MS/MS by isolating and dissociating each compound in a four component mixture of tetraalkylphosphonium cations. We also discuss the optimization of collision energy and the length of time that the SID target is available for collision, two parameters that are important in the performance of these experiments.

Absorption mode Fourier transform electrostatic linear ion trap mass spectrometry.

In Fourier transform mass spectrometry, it is well-known that plotting the spectrum in absorption mode rather than magnitude mode has several advantages. However, magnitude spectra remain commonplace due to difficulties associated with determining the phase of each frequency at the onset of data acquisition, which is required for generating absorption spectra. The phasing problem for electrostatic traps is much simpler than for Fourier transform ion cyclotron resonance (FTICR) instruments, which greatly simplifies the generation of absorption spectra. Here, we present a simple method for generating absorption spectra from a Fourier transform electrostatic linear ion trap mass spectrometer. The method involves time shifting the data prior to Fourier transformation in order to synchronize the onset of data acquisition with the moment of ion acceleration into the electrostatic trap. Under these conditions, the initial phase of each frequency at the onset of data acquisition is zero. We demonstrate that absorption mode provides a 1.7-fold increase in resolution (full width at half maximum, fwhm) as well as reduced peak tailing. We also discuss methodology that may be applied to unsynchronized data in order to determine the time shift required to generate an absorption spectrum.

Nondestructive tandem mass spectrometry using a linear quadrupole ion trap coupled to a linear electrostatic ion trap.

A novel hybrid tandem mass spectrometer is presented that combines a linear quadrupole ion trap (QLIT) with a linear electrostatic ion trap (ELIT), which is composed of opposing ion mirrors. The QLIT is used both as an accumulation device for the pulsed injection of ions into the ELIT and as a collision cell for ions released from the ELIT and back into the QLIT. Ions are subjected to mass analysis in the ELIT via Fourier transformation of the time-domain signal obtained from an image current measurement using a pick-up electrode in the field-free region of the ELIT. The nondestructive nature of ion detection and the relatively straightforward axial entrance and exit of ions into and from the ELIT allow for the execution of nondestructive tandem mass spectrometry experiments whereby both the initial mass spectrum and the product ion spectrum are obtained on the same initial ion population. The timed pulsing of a deflection electrode, in conjunction with the release of ions from the ELIT, allows for the selection of precursor ions for recapture by the QLIT. The transfer of ions back and forth between the QLIT and ELIT is illustrated with Cs ions, the selection of precursor ions is demonstrated with isotopes of tetraoctylammonium cations, and complete nondestructive tandem mass spectrometry experiments are demonstrated with a mixture of angiotensin II and bradykinin cations. With the current apparatus, the efficiency for the process of recapturing ions and then reinjecting them into the ELIT is 35%-40%. The instrument is capable of isolating an ion from a neighbor with a mass as close as 1 part in 500, with negligible loss of the desired species.

The effect of reagent charge state on the charge inversion efficiency of singly charged polyatomic ions in the gas phase.

A variety of combinations of oppositely charged ions have been reacted to examine the role of the charge state from a multiply protonated or multiply deprotonated reagent ion on the efficiency of conversion of a singly charged ion of opposite polarity to a singly charged ion of the same polarity as the reagent. Maximum efficiencies on the order of tens of percent were observed. A threshold for charge inversion was noted in all cases and, with one exception, a clear decrease in efficiency was also noted at high charge states. A model was developed to predict charge inversion efficiency based on charge states, cross-sections of the reactants, and relevant thermodynamic ion affinity values for the reactants and products. The model predicts a threshold for charge inversion, although the prediction does not match the observed threshold quantitatively. This discrepancy is likely due to a simplifying assumption that is not justified on a quantitative basis but which does reproduce the qualitative trend. The model does not predict the major decrease in efficiency at high charge states. However, calculations show that the kinetic energies of the charge inversion products can lead to significant scattering losses at high charge states of the ion-ion collision complex.

A mechanical nanomembrane detector for time-of-flight mass spectrometry.

We describe here a new principle for ion detection in time-of-flight (TOF) mass spectrometry in which an impinging ion packet excites mechanical vibrations in a silicon nitride (Si(3)N(4)) nanomembrane. The nanomembrane oscillations are detected by means of time-varying field emission of electrons from the mechanically oscillating nanomembrane. Ion detection is demonstrated in the MALDI-TOF analysis of proteins varying in mass from 5729 (insulin) to 150,000 (Immunoglobulin G) daltons. The detector response agrees well with the predictions of a thermomechanical model in which the impinging ion packet causes a nonuniform temperature distribution in the nanomembrane, exciting both fundamental and higher order oscillations.

Parallel detection of intrinsic fluorescence from peptides and proteins for quantification during mass spectrometric analysis.

Direct mass spectrometric quantification of peptides and proteins is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describe here the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins just prior to their exit and electrospray ionization from an ESI capillary. The fluorescence signal provides a quantifiable measure of the amount of protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS) on the ESI-generated ions provides information on identity. We fabricated an inexpensive, modular fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a ∼300 nL fluorescence detection cell integrated into the fused-silica separation column. The fluorescence signal is linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range. Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa.

Controlling charge on levitating drops.

Levitation technologies are used in containerless processing of materials, as microscale manipulators and reactors, and in the study of single drops and particles. Presented here is a method for controlling the amount and polarity of charge on a levitating drop. The method uses single-axis acoustic levitation to trap and levitate a single, initially neutral drop with a diameter between 400 microm and 2 mm. This drop is then charged in a controllable manner using discrete packets of charge in the form of charged drops produced by a piezoelectric drop-on-demand dispenser equipped with a charging electrode. The magnitude of the charge on the dispensed drops can be adjusted by varying the voltage applied to the charging electrode. The polarity of the charge on the added drops can be changed allowing removal of charge from the trapped drop (by neutralization) and polarity reversal. The maximum amount of added charge is limited by repulsion of like charges between the drops in the trap. This charging scheme can aid in micromanipulation and the study of charged drops and particles using levitation.