"Magic" Ionization Mass Spectrometry

Abstract

The systematic study of the temperature and pressure dependence of matrix-assisted ionization (MAI) led us to the discovery of the seemingly impossible, initially explained by some reviewers as either sleight of hand or the misinterpretation by an overzealous young scientist of results reported many years before and having little utility. The “magic” that we were attempting to report was that with matrix assistance, molecules, at least as large as bovine serum albumin (66 kDa), are lifted into the gas phase as multiply charged ions simply by exposure of the matrix:analyte sample to the vacuum of a mass spectrometer. Applied heat, a laser, or voltages are not necessary to achieve charge states and ion abundances only previously observed with electrospray ionization (ESI). The fundamentals of how solid phase volatile or nonvolatile compounds are converted to gas-phase ions without added energy currently involves speculation providing a great opportunity to rethink mechanistic understanding of ionization processes used in mass spectrometry. Improved understanding of the mechanism(s) of these processes and their connection to ESI and matrix-assisted laser desorption/ionization may provide opportunities to further develop new ionization strategies for traditional and yet unforeseen applications of mass spectrometry. This Critical Insights article covers developments leading to the discovery of a seemingly magic ionization process that is simple to use, fast, sensitive, robust, and can be directly applied to surface characterization using portable or high performance mass spectrometers.

Graphical Abstract

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Scheme 1

MAI matrix structures indicating general categories for mode of operation: *work with the assistance of higher inlet temperatures without or with a laser, **work with a laser and the assistance of pressure, ***work with the assistance of pressure in the absence of a laser

Scheme 2

“A phase diagram from a mass spectrometry perspective” illustrating commonalities of the transitions from solid and liquid states to the desired gas-phase ions for use in mass spectrometry by the new ionization process governed by temperature and pressure relative to traditional ionization methods using high voltages and sometimes a laser. A matrix can be a solid or a solvent

Figure 1

Mass spectra of ubiquitin (MW8560) acquired using (a) MAII with 2,2'-azobis(2-methylpropionitrile) 11 matrix acidified with acetic acid, (b) MAII with anthracene 14 matrix in ACN:water acidified with 1% hydrochloric acid, (c) SAII (no matrix added to solution), and (d) dissolved MAII with 2,2'-azobis(2-methylpropane) 12 added into the ubiquitin solution acquired on the LTQ Velos mass spectrometer at 450°C inlet capillary temperature. Red numbers indicate the charge states, and blue numbers in the upper right corner provide relative ion abundance. Modified from Figures 3 and 6, with permission from Li et al. [19]

Figure 2

LSIV mass spectra: (top half) N-acetylated myelin basic protein fragment (MBP, MW 1833) with 2,5-DHAP 6 matrix acquired using the Waters SYNAPT G2 mass spectrometer intermediate pressure MALDI ion source. (a) ESI tune, sample plate 0 V, extractor lens 10 V, hexapole bias 10 V, and laser power of 5 J/cm², and (b) MALDI tune, sample plate 20 V, hexapole bias 10 V, extractor lens 10 V, and a laser power of 15 J/cm². (Bottom half) (c) carbonic anhydrase (MW 29 kDa) and (d) MBP peptide prepared using the dried droplet method with 2-NPG 18 and acquired in reflectron mode on Bruker high vacuum MALDI-TOF/TOF mass spectrometer. Red numbers indicate the charge states, and blue numbers in the upper right corner provide relative ion abundance. Modified from Figures 1, 4, and Supplementary S11, with permission from Trimpin et al. [20]

Figure 3

(a) LSII-MS and (b) MAII-MS spectra of 20 pmol of BSA (~66 kDa) using 2-NPG 18 matrix and a 200°C inlet capillary on an LTQ-Velos mass spectrometer. The ion trap was set to 10 microscans and a 100 ms max injection time. The starred and labeled peaks are believed to be the protonated multiply charged protein ions. Red numbers and asterisks indicate the charge states. Modified from Figure 2, with permission from Lietz et al. [94]

Figure 4

MAIV-MS of lysozyme, 14.3 kDa (a) total ion chronogram and (b) mass spectrum; (c) MAIV mass spectrum of bovine serum albumin, 66 kDa, acquired on an intermediate pressure MALDI source of a SYNAPT G2 with the laser off using the matrix 3-NBN 22. Red numbers and asterisks indicate the charge states, and blue numbers in the upper right corner of each spectrum provide relative ion abundance. Modified from Figure 1, with permission from Inutan et al. [16]

Scheme 3

Acronyms of methods used in MS for analysis of nonvolatile compounds shown on the branches of a tree and its reflection. Matrix-assisted ionization (MAI) is shown as the base of the tree. Methods that operate from solution (left) and solid states (right) are organized from bottom to top into hot (light red) to cold (blue) energetic conditions with ionization methods that produce multiple (top) or primarily singly (bottom) charged ions. Left top to bottom: sonic spray ionization (SSI), desorption electrospray ionization (DESI), electrospray ionization (ESI), electrospray ionization inlet (ESII), solvent-assisted ionization inlet (SAII), dissolved matrix-assisted ionization inlet (d-MAII), thermospray ionization (TSI), fast atom bombardment (FAB); right top to bottom: cold SAII (c-SAII), matrix-assisted ionization vacuum (MAIV), matrix-assisted ionization inlet (MAII), laserspray ionization inlet (LSII), laserspray ionization vacuum (LSIV), atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI), pulse-heating ionization (PHI), and MALDI