Beixi Wang

A Mechanism for Ionization of Nonvolatile Compounds in Mass Spectrometry: Considerations from MALDI and Inlet Ionization

Mechanistic arguments relative to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) address observations that predominately singly charged ions are detected. However, recently a matrix assisted laser ablation method, laserspray ionization (LSI), was introduced that can use the same sample preparation and laser as MALDI, but produce highly charged ions from proteins. In MALDI, ions are generated from neutral molecules by the photon energy provided to a matrix, while in LSI ions are produced inside a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer. Some LSI matrices also produce highly charged ions with MALDI ion sources operated at intermediate pressure or high vacuum. The operational similarity of LSI to MALDI, and the large difference in charge states observed by these methods, provides information of fundamental importance to proposed ionization mechanisms for LSI and MALDI. Here, we present data suggesting that the prompt and delayed ionization reported for vacuum MALDI are both fast processes relative to producing highly charged ions by LSI. The energy supplied to produce these charged clusters/droplets as well as their size and time available for desolvation are determining factors in the charge states of the ions observed. Further, charged droplets/clusters may be a common link for ionization of nonvolatile compounds by a variety of MS ionization methods, including MALDI and LSI.

Commercial Intermediate Pressure MALDI Ion Mobility Spectrometry Mass Spectrometer Capable of Producing Highly Charged Laserspray Ionization Ions

The first examples of highly charged ions observed under intermediate pressure (IP) vacuum conditions are reported using laser ablation of matrix/analyte mixtures. The method and results are similar to those obtained at atmospheric pressure (AP) using laserspray ionization (LSI) and/or matrix assisted inlet ionization (MAII). Electrospray ionization (ESI), LSI, and MAII are methods operating at AP and have been shown, with or without the use of a voltage or a laser, to produce highly charged ions with very similar ion abundance and charge states. A commercial matrix-assisted laser desorption/ionization ion mobility spectrometry (IMS) mass spectrometry (MS) instrument (SYNAPT G2) was used for the IP developments. The necessary conditions for producing highly charged ions of peptides and small proteins at IP appear to be a pressure drop region and the use of suitable matrixes and laser fluence. Ionization to produce these highly charged ions under the low pressure conditions of IP does not require specific heating or a special inlet ion transfer region. However, under the current setup, ubiquitin is the highest molecular weight protein observed. These findings are in accord with the need to provide thermal energy in the pressure drop region, similar to LSI and MAII, to improve sensitivity and extend the types of compounds that produce highly charged ions. The practical utility of IP-LSI in combination with IMS-MS is demonstrated for the analysis of model mixtures composed of a lipid, peptides, and a protein. Further, endogenous multiply charged peptides are observed directly from delipified mouse brain tissue with drift time distributions that are nearly identical in appearance to those obtained from a synthesized neuropeptide standard analyzed by either LSI- or ESI-IMS-MS at AP. Efficient solvent-free gas-phase separation enabled by the IMS dimension separates the multiply charged peptides from lipids that remained on the delipified tissue. Lipid and peptide families are exceptionally well separated because of the ability of IP-LSI to produce multiple charging.

Extending the Laserspray Ionization Concept to Produce Highly Charged Ions at High Vacuum on a Time-of-Flight Mass Analyzer

A new matrix compound, 2-nitrophloroglucinol, is reported which not only produces highly charged ions similar to electrospray ionization (ESI) under atmospheric pressure (AP) and intermediate pressure (IP) laserspray ionization (LSI) conditions but also the most highly charged ions so far observed for small proteins in mass spectrometry (MS) under high vacuum (HV) conditions. This new matrix extends the compounds that can successfully be employed as matrixes with LSI, as demonstrated on an LTQ Velos (Thermo) at AP, a matrix-assisted laser desorption/ionization (MALDI)-ion mobility spectrometry (IMS) time-of-flight (TOF) SYNAPT G2 (Waters) at IP, and MALDI-TOF Ultraflex, UltrafleXtreme, and Autoflex Speed (Bruker) mass spectrometers at HV. Measurements show that stable multiple charged molecular ions of proteins are formed under all pressure conditions indicating softer ionization than MALDI, which suffers a high degree of metastable fragmentation when multiply charged ions are produced. An important analytical advantage of this new LSI matrix are the potential for high sensitivity equivalent or better than AP-LSI and vacuum MALDI and the potential for enhanced mass selected fragmentation of the abundant highly charged protein ions. A second new LSI matrix, 4,6-dinitropyrogallol, produces abundant multiply charged ions at AP but not under HV conditions. The differences in these similar compounds ability to produce multiply charged ions under HV conditions is believed to be related to their relative ability to evaporate from charged matrix/analyte clusters.

New ionization processes and applications for use in mass spectrometry.

Abstract Mass spectrometry (MS) continues to improve at a rapid pace as most prominently witnessed for mass analyzers and fragmentation technology. Ionization methods have also seen resurgence with ambient ionization approaches gaining a foothold because they often provide a convenient and direct means of sample analysis. Nevertheless, a vast majority of biological analyses using MS apply electrospray ionization or matrix-assisted laser desorption/ionization, methods introduced in the 1980s, or variants thereof. To further advance applications by MS such as protein characterization, and, for example, addressing their location within specific cell types, the progress in mass analyzer and fragmentation technology needs to be matched with similar advances in ionization technology. It is imperative to seek ionization methods that more efficiently convert molecules, to gas-phase ions in a way that allows high transfer efficiency to the mass analyzer and subsequently the detector to achieve a more complete picture of sample composition. This review provides a snapshot of fundamental aspects of new ionization methods and potential biological and medical applications.

Producing highly charged ions without solvent using laserspray ionization: a total solvent-free analysis approach at atmospheric pressure.

First examples of highly charged ions in mass spectrometry (MS) produced from the solid state without using solvent during either sample preparation or mass measurement are reported. Matrix material, matrix/analyte homogenization time and frequency, atmospheric pressure (AP) to vacuum inlet temperature, and mass analyzer ion trap conditions are factors that influence the abundance of the highly charged ions created by laserspray ionization (LSI). LSI, like matrix-assisted laser desorption/ionization (MALDI), uses laser ablation of a matrix/analyte mixture from a surface to produce ions. Preparing the matrix/analyte sample without the use of solvent provides the ability to perform total solvent-free analysis (TSA) consisting of solvent-free ionization and solvent-free gas-phase separation using ion mobility spectrometry (IMS) MS. Peptides and small proteins such as non-β-amyloid components of Alzheimers disease and bovine insulin are examples in which LSI and TSA were combined to produce multiply charged ions, similar to electrospray ionization, but without the use of solvent. Advantages using solvent-free LSI and IMS-MS include simplicity, rapid data acquisition, reduction of sample complexity, and the potential for an enhanced effective dynamic range. This is achieved by more inclusive ionization and improved separation of mixture components as a result of multiple charging.

High-Throughput Solvent Assisted Ionization Inlet for Use in Mass Spectrometry

In this work we developed a multiplexed analysis platform providing a simple high-throughput means to characterize solutions. Automated analyses, requiring less than 5 s per sample without carryover and 1 s per sample, accepting minor cross contamination, was achieved using multiplexed solvent assisted ionization inlet (SAII) mass spectrometry (MS). The method involves sequentially moving rows of pipet tips containing sample solutions in close proximity to the inlet aperture of a heated mass spectrometer inlet tube. The solution is pulled from the container into the mass spectrometer inlet by the pressure differential at the mass spectrometer inlet aperture. This sample introduction method for direct injection of solutions is fast, easily implemented, and widely applicable, as is shown by applications ranging from small molecules to proteins as large as carbonic anhydrase (molecular weight ca. 29,000). MS/MS fragmentation is applicable for sample characterization. An x,y-stage and common imaging software are incorporated to map the location of components in the sample wells of a microtiter plate. Location within an x,y-array of different sample solutions and the relative concentration of the sample are displayed using ion intensity maps.

High-throughput characterization of small and large molecules using only a matrix and the vacuum of a mass spectrometer.

Matrix assisted ionization vacuum (MAIV) rapidly generates gas-phase analyte ions from subliming solid-phase matrix:analyte crystals for analysis by mass spectrometry (MS). Ionization from the solid-phase allows the use of a variety of surfaces for introducing matrix:analyte samples to the vacuum of a mass spectrometer, including common laboratory materials, such as disposable pipet tips, filter paper, tooth picks, and nylon mesh. MAIV is shown here to be capable of analyses as fast as 3 s per sample with achievable sensitivities in the low femtomole range. MAIV-MS coupled with ion mobility spectrometry (IMS)-MS and tandem mass spectrometry (MS/MS) is shown to be especially powerful for analysis and characterization of a wide range of molecules ranging from small molecules such as drugs and metabolites (∼300 Da) to intact proteins (25.6 kDa). Automated sample introduction is demonstrated on two different commercial mass spectrometers using a programmable XYZ stage. A MAIV high-throughput nontargeted MS(E) approach is also demonstrated utilizing IMS for rapid characterization of small molecules and peptides from standard solutions, as well as drug spiked human urine.

Matrix-Assisted Ionization Vacuum for High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometers

Matrix-assisted ionization vacuum (MAIV) produces charge states similar to electrospray ionization (ESI) from the solid state without requiring high voltage or added heat. MAIV differs from matrix-assisted laser desorption/ionization (MALDI) in that no laser is needed and abundant multiply charged ions are produced from molecules having multiple basic sites such as proteins. Here we introduce simple modifications to the commercial vacuum MALDI and ESI sources of a 9.4 T Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer to perform MAIV from both intermediate and atmospheric pressure. The multiply charged ions are shown for the proteins bovine insulin, ubiquitin, and lysozyme using 3-nitrobenzonitrile as matrix. These are the first examples of MAIV operating at pressures as low as 10(-6) mbar in an FT-ICR mass spectrometer source, and the expected mass resolving power of 100000 to 400000 is achieved. Identical protein charge states are observed with and without laser ablation indicating minimal, if any, role of photochemical ionization for the compounds studied.

Gas-Phase Ions Produced by Freezing Water or Methanol for Analysis Using Mass Spectrometry

Introducing water or methanol containing a low concentration of volatile or nonvolatile analyte into an inlet tube cooled with dry ice linking atmospheric pressure and the first vacuum stage of a mass spectrometer produces gas-phase ions even of small proteins that can be detected by mass spectrometry. Collision-induced dissociation experiments conducted in the first vacuum region of the mass spectrometer suggest analyte ions being protected by a solvent cage. The charges may be produced by processes similar to those proposed for charge separation under freezing conditions in thunderclouds. By this process, the surface of an ice pellet is charged positive and the interior negative so that removal of surface results in charge separation. A reversal of surface charge is expected for a heated droplet surface, and this is observed by heating rather than cooling the inlet tube. These observations are consistent with charged supercooled droplets or ice particles as intermediates in the production of analyte ions under freezing conditions.

Characterizing synthetic polymers and additives using new ionization methods for mass spectrometry

RATIONALE New inlet and vacuum ionization methods provide advantages of specificity, simplicity and speed for the analysis of synthetic polymers and polymer additives directly from surfaces such as fibers using mass spectrometry (MS) on different commercial mass spectrometers (Waters SYNAPT G2, Thermo LTQ Velos). METHODS We compare inlet ionization methods with the recently discovered vacuum ionization method. This method, termed matrix assisted ionization vacuum (MAIV), utilizes the matrix 3-nitrobenzonitrile (3-NBN) for the analysis of synthetic polymers and additives without additional energy input by simply exposing the matrix:analyte:salt to the vacuum of the mass spectrometer. Matrix:analyte:salt samples can be introduced while dry (surfaces, e.g. glass slides, pipet tips) or slightly wet (e.g. filter paper, pipet tips). RESULTS Compounds ionized by these methods can be analyzed in both positive and negative detection modes through cationization or deprotonation, respectively. The dynamic range of the experiment can be enhanced, as well as structural analysis performed, by coupling the vacuum ionization method with ion mobility spectrometry mass spectrometry (IMS-MS) and tandem mass spectrometric (MS/MS) fragmentation. CONCLUSIONS The specificity of 3-NBN matrix to ionize small and large nonvolatile analyte molecules by MAIV makes this matrix a good choice for observing low-abundance additives in the presence of large amounts of synthetic polymer using MS.