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1. An overview of biological applications and fundamentals of new inlet and vacuum ionization technologies

   https://doi.org/10.1002/rcm.8829  

   Trimpin, Sarah ; Marshall, Darrell D. ; Karki, Santosh ; Madarshahian, Sara ; Hoang, Khoa ; Meher, Anil K. ; Pophristic, Milan ; Richards, Alicia L. ; Lietz, Christopher B. ; Fischer, Joshua L. ; et al ( November 2020 , Rapid Communications in Mass Spectrometry)

   The developments of new ionization technologies based on processes previously unknown to mass spectrometry (MS) have gained significant momentum. Herein we address the importance of understanding these unique ionization processes, demonstrate the new capabilities currently unmet by other methods, and outline their considerable analytical potential.

   Theinletandvacuumionization methods of solvent‐assisted ionization (SAI), matrix‐assisted ionization (MAI), and laserspray ionization can be used with commercial and dedicated ion sources producing ions from atmospheric or vacuum conditions for analyses of a variety of materials including drugs, lipids, and proteins introduced from well plates, pipet tips and plate surfaces with and without a laser using solid or solvent matrices. Mass spectrometers from various vendors are employed.

   Results are presented highlighting strengths relative to ionization methods of electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization. We demonstrate the utility of multi‐ionization platforms encompassing MAI, SAI, and ESI and enabling detection of what otherwise is missed, especially when directly analyzing mixtures. Unmatched robustness is achieved with dedicated vacuum MAI sources with mechanical introduction of the sample to the sub‐atmospheric pressure (vacuumMAI). Simplicity and use of a wide array of matrices are attained using a conduit (inletionization), preferably heated, with sample introduction from atmospheric pressure. Tissue, whole blood, urine (including mouse, chicken, and human origin), bacteria strains and chemical on‐probe reactions are analyzed directly and, especially in the case ofvacuumionization, without concern of carryover or instrument contamination.

   Examples are provided highlighting the exceptional analytical capabilities associated with the novel ionization processes in MS that reduce operational complexity while increasing speed and robustness, achieving mass spectra with low background for improved sensitivity, suggesting the potential of this simple ionization technology to drive MS into areas currently underserved, such as clinical and medical applications.

    

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2. Development of a robotics platform for automated multi‐ionization mass spectrometry

   https://doi.org/10.1002/rcm.8449  

   Karki, Santosh ; Meher, Anil K. ; Inutan, Ellen D. ; Pophristic, Milan ; Marshall, Darrell D. ; Rackers, Kevin ; Trimpin, Sarah ; McEwen, Charles N. ( June 2019 , Rapid Communications in Mass Spectrometry)

   Successful coupling of a multi‐ionization automated platform with commercially available mass spectrometers provides improved coverage of compounds in complex mixtures through implementation of new and traditional ionization methods. The versatility of the automated platform is demonstrated through coupling with mass spectrometers from two different vendors. Standards and complex biological samples were acquired using electrospray ionization (ESI), solvent‐assisted ionization (SAI) and matrix‐assisted ionization (MAI).

   The MS™ prototype automated platform samples from 96‐ or 384‐well plates as well as surfaces. The platform interfaces with Thermo Fisher Scientific mass spectrometers by replacement of the IonMax source, and on Waters mass spectrometers with additional minor source inlet modifications. The sample is transferred to the ionization region using a fused‐silica or metal capillary which is cleaned between acquisitions using solvents. For ESI and SAI, typically 1 μL of sample solution is drawn into the capillary tube and for ESI slowly dispensed near the inlet of the mass spectrometer with voltage placed on the delivering syringe barrel to which the tubing is attached, while for SAI the sample delivery tubing inserts into the inlet without the need for high voltage. For MAI, typically, 0.2 μL of matrix solution is drawn into the syringe before drawing 0.1 μL of the sample solution and dispensing to dry before insertion into the inlet.

   A comparison study of a mixture of angiotensin I, verapamil, crystal violet, and atrazine representative of peptides, drugs, dyes, and herbicides using SAI, MAI, and ESI shows large differences in ionization efficiency of the various components. Solutions of a mixture of erythromycin and azithromycin in wells of a 384‐microtiter well plate were mass analyzed at the rate ofca1 min per sample using MAI and ESI. In addition, we report the analysis of bacterial extracts using automated MAI and ESI methods. Finally, the ability to perform surface analysis with the automated platform is also demonstrated by directly analyzing dyes separated on a thin‐layer chromatography (TLC) plate and compounds extracted from the surface of a beef liver tissue section.

   The prototype multi‐ionization automated platform offers solid matrix introduction used with MAI, as well as solution introduction using either ESI or SAI. The combination of ionization methods extends the types of compounds which are efficiently ionized and is especially valuable with complex mixtures as demonstrated for bacterial extracts. While coupling of the automated multi‐ionization platform to Thermo and Waters mass spectrometers is demonstrated, it should be possible to interface it with most commercial mass spectrometers.

    

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3. A Combination MAI and MALDI Vacuum Source Operational from Atmospheric Pressure for Fast, Robust, and Sensitive Analyses

   https://doi.org/10.1021/jasms.0c00298  

   Hoang, Khoa. ; Trimpin, Sarah ; McEwen, Charles N. ; Pophristic, Milan ( January 2020 , Journal of the American Society for Mass Spectrometry)

   Previously, vacuum matrix-assisted ionization (vMAI) was employed with matrix:analyte sample introduction into the vacuum of the mass spectrometer on a probe sample introduction device. Low attomole detection was achieved while no carryover was observed even for concentrated samples. Here, we report a new vacuum ionization source designed to duplicate the sensitivity and robustness of probe device while providing fast multi-sample introduction to vacuum and rapid sequential ionization. Exposure of a sample to the vacuum of the mass spectrometer provides spontaneous ionization of volatile as well as nonvolatile analytes without the need of external energy input. However, the novel source design described herein, in addition to vMAI, can employ a laser to obtain vacuum matrix-assisted laser desorption/ionization (vMALDI). In particular, ionization by vMAI or vMALDI is achieved by using the appropriate matrix. Switching between ionization modes is accomplished in a few seconds. We present results demonstrating the utility of the two ionization methods in combination to improve molecular analyses of sample composition. In both ionization modes, multiple samples can be sequentially and rapidly acquired to increase throughput in MS. With the prototype source, samples were acquired in as little as 1 second per sample. Exchanging multi-sample plates can be accomplished in as little as 2 seconds suggesting low-cost high throughput automation when properly developed. 

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4. Comparison of gaseous ubiquitin ion structures obtained from a solid and solution matrix using ion mobility spectrometry/mass spectrometry

   https://doi.org/10.1002/rcm.8793  

   Inutan, Ellen D. ; Jarois, Dean R. ; Lietz, Christopher B. ; El‐Baba, Tarick J. ; Elia, Efstathios A. ; Karki, Santosh ; Sampat, Andjoe A. S. ; Foley, Casey D. ; Clemmer, David E. ; Trimpin, Sarah ( August 2020 , Rapid Communications in Mass Spectrometry)

   Examining surface protein conformations, and especially achieving this with spatial resolution, is an important goal. The recently discovered ionization processes offer spatial‐resolution measurements similar to matrix‐assisted laser desorption/ionization (MALDI) and produce charge states similar to electrospray ionization (ESI) extending higher‐mass protein applications directly from surfaces on high‐performance mass spectrometers. Studying a well‐interrogated protein by ion mobility spectrometry‐mass spectrometry (IMS‐MS) to access effects on structures using a solidvs.solvent matrix may provide insights.

   Ubiquitin was studied by IMS‐MS using new ionization processes with commercial and homebuilt ion sources and instruments (Waters SYNAPT G2(S)) and homebuilt 2 m drift‐tube instrument; MS™ sources). Mass‐to‐charge and drift‐time (t_d)‐measurements are compared for ubiquitin ions obtained byinletandvacuumionization using laserspray ionization (LSI), matrix‐ (MAI) and solvent‐assisted ionization (SAI), respectively, and compared with those from ESI under conditions that are most comparable.

   Using the same solution conditions with SYNAPT G2(S) instruments, t_d‐distributions of various ubiquitin charge states from MAI, LSI, and SAI are similar to those from ESI using a variety of solvents, matrices, extraction voltages, a laser, and temperature only, showing subtle differences in more compact features within the elongated distribution of structures. However, on a homebuilt drift‐tube instrument, within the elongated distribution of structures, both similar and different t_d‐distributions are observed for ubiquitin ions obtained by MAI and ESI. MAI‐generated ions are frequently narrower in their t_d‐distributions.

   Direct comparisons between ESI and the new ionization methods operational directly from surfaces suggest that the protein in its solution structure prior to exposure to the ionization event is either captured (frozen out) at the time of crystallization, or that the protein in the solid matrix is associated with sufficient solvent to maintain the solution structure, or, alternatively, that the observed structures are those related to what occurs in the gas phase with ESI‐ or MAI‐generated ions and not with the solution structures.

    

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5. Toward understanding the ionization mechanism of matrix‐assisted ionization using mass spectrometry experiment and theory

   https://doi.org/10.1002/rcm.8382  

   Lee, Chuping ; Inutan, Ellen D. ; Chen, Jien Lian ; Mukeku, Mutanu M. ; Weidner, Steffen M. ; Trimpin, Sarah ; Ni, Chi‐Kung ( March 2019 , Rapid Communications in Mass Spectrometry)

   Matrix‐assisted ionization (MAI) mass spectrometry does not require voltages, a laser beam, or added heat to initiate ionization, but it is strongly dependent on the choice of matrix and the vacuum conditions. High charge state distributions of nonvolatile analyte ions produced by MAI suggest that the ionization mechanism may be similar to that of electrospray ionization (ESI), but different from matrix‐assisted laser desorption/ionization (MALDI). While significant information is available for MAI using mass spectrometers operating at atmospheric and intermediate pressure, little is known about the mechanism at high vacuum.

   Eleven MAI matrices were studied on a high‐vacuum time‐of‐flight (TOF) mass spectrometer using a 266 nm pulsed laser beam under otherwise typical MALDI conditions. Detailed comparisons with the commonly used MALDI matrices and theoretical prediction were made for 3‐nitrobenzonitrile (3‐NBN), which is the only MAI matrix that works well in high vacuum when irradiated with a laser.

   Screening of MAI matrices with good absorption at 266 nm but with various degrees of volatility and laser energies suggests that volatility and absorption at the laser wavelength may be necessary, but not sufficient, criteria to explain the formation of multiply charged analyte ions. 3‐NBN produces intact, highly charged ions of nonvolatile analytes in high‐vacuum TOF with the use of a laser, demonstrating that ESI‐like ions can be produced in high vacuum. Theoretical calculations and mass spectra suggest that thermally induced proton transfer, which is the major ionization mechanism in MALDI, is not important with the 3‐NBN matrix at 266 nm laser wavelength. 3‐NBN:analyte crystal morphology is, however, important in ion generation in high vacuum.

   The 3‐NBN MAI matrix produces intact, highly charged ions of nonvolatile compounds in high‐vacuum TOF mass spectrometers with the aid of ablation and/or heating by laser irradiation, and shows a different ionization mechanism from that of typical MALDI matrices.

    

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