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

Abstract

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.

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

“…The nearly identical charge states produced in IP-LSI relative to AP-LSI using 2-NPG or 2,5-DHAP matrices for peptides and small proteins [42] suggests that photoionization is not responsible for multiply charged ions produced under IP conditions. The operational similarity of IP-LSI and MALDI further suggests that photoionization may also not be a strong contributor to cluster ionization in MALDI.…”

“…The commonly used UV MALDI matrix, 2,5-dihydroxyacetophenone (2,5-DHAP), produces nearly identical highly charged ions from laser ablation initiated at AP or IP (Scheme S1D) [42]. Because AP and IP results for LSI can have nearly identical charge state distributions using the same matrix (2,5-DHAP)/analyte mixture [42,43], we assume that ionization in this case is governed by similar mechanisms. In AP-LSI, the function of the laser is to ablate the matrix/analyte mixture in a way that some of the ablated material enters the entrance aperture of the mass spectrometer.…”

“…A Waters SYNAPT G2 mass spectrometer (Manchester, UK) equipped with a commercial MALDI source and operated with a Nd:YAG laser (355 nm, 200 Hz) was used to carry-out IP-LSI as previously described [42]. Briefly, acquisitions were performed in positive ion sensitivity mode using the following LSI settings: 0 V on the sample plate holder, 10 V "extraction," 10 V "hexapole bias," and 5 V "aperture 0."…”

“…By such mechanisms, particle or droplet contact with a surface could initiate charging ( Figure S7). However, the finding that nearly equal ion abundances and charge states are produced using different materials for inlet tube construction, and the recent demonstration that laser ablation of 2,5-DHAP [42] and 2-NPG [45] under IP conditions produce highly charged ion distributions nearly identical to those seen in AP-LSI are not easily reconciled with a purely surface charging mechanism. An alternative ionization possibility for matrix charging that can operate at AP and IP is statistical charging by an explosive fracturing mechanism, including such processes as bubble bursting.…”

Improving the Sensitivity of Matrix-Assisted Ionization (MAI) Mass Spectrometry Using Ammonium Salts

“…In MAI, gasphase analyte ions are produced when the analyte in a small molecule matrix compound is exposed to heat [6] and/or the vacuum of the mass spectrometer [2,3]. Laserspray ionization inlet (LSII) [7] and vacuum (LSIV) [8] are subsets of MAI in which a laser ablates matrix/analyte particles/droplets into the vacuum of the mass spectrometer where ionization occurs, presumably by a similar mechanism as MAI [9]. Solventassisted ionization inlet (SAII) [10] can be considered a MAI method in which the solvent serves as a matrix allowing solution introduction into the inlet by e.g.…”