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.

Direct analysis of samples using atmospheric pressure ionization (API) provides a more rapid method for analysis of volatile and semivolatile compounds than vacuum solids probe methods and can be accomplished on commercial API mass spectrometers. With only a simple modification to either an electrospray (ESI) or atmospheric pressure chemical ionization (APCI) source, solid as well as liquid samples can be analyzed in seconds. The method acts as a fast solids/liquid probe introduction as well as an alternative to the new direct analysis in real time (DART) and desorption electrospray ionization (DESI) methods for many compound types. Vaporization of materials occurs in the hot nitrogen gas stream flowing from an ESI or APCI probe. Ionization of the thermally induced vapors occurs by corona discharge under standard APCI conditions. Accurate mass and mass-selected fragmentation are demonstrated as is the ability to obtain ions from biological tissue, currency, and other objects placed in the path of the hot nitrogen stream.

The first example of a matrix-assisted laser desorption/ionization (MALDI) process producing multiply charged mass spectra nearly identical to those observed with electrospray ionization (ESI) is presented. MALDI is noted for its ability to produce singly charged ions, but in the experiments described here multiply charged ions are produced by laser ablation of analyte incorporated into a common MALDI matrix, 2,5-dihydroxybenzoic acid, using standard solvent-based sample preparation protocols. Laser ablation is known to produce matrix clusters in MALDI provided a threshold energy is achieved. We propose that these clusters (liquid droplets) are highly charged, and under conditions that produce sufficient matrix evaporation, ions are field-evaporated from the droplets similarly to ESI. Because of the multiple charging, advanced mass spectrometers with limited mass-to-charge range can be used for protein characterization. Thus, using an Orbitrap mass spectrometer, low femtomole quantities of proteins produce full-range mass spectra at 100,000 mass resolution with <5-ppm mass accuracy and with 1-s acquisition. Furthermore, the first example of protein fragmentation using electron transfer dissociation with MALDI is presented.

A new inlet ionization method requiring no voltage or laser, and using water, methanol, or water/organic solvent mixtures, is shown to produce mass spectra similar to those obtained with electrospray ionization (ESI) for small molecules, peptides, and proteins, at least as large as carbonic anhydrase, with sensitivity that surpasses ESI. With the use of wide mass range acquisitions at 100,000 mass resolution on an Orbitrap Exactive, detection limits below parts per trillion are obtained for small molecules such as arginine, ciprofloxacin, and acetaminophen. Low attomoles of bovine insulin consumed produced a multiply charged mass spectrum. Ions are generated, even using pure water as solvent, within the heated inlet tube linking atmospheric pressure with the first vacuum stage of the Orbitrap Exactive. The extremely high sensitivity observed at this early stage of solvent assisted inlet ionization (SAII) development suggests that inlet ionization may surpass nanoelectrospray in sensitivity but without the need for extremely low solvent flows.

Laserspray ionization (LSI) is a new approach to producing multiply charged ions from solids on surfaces by laser ablation of matrixes commonly used in matrix-assisted laser desorption/ionization (MALDI). We show that the only necessity of the laser for producing multiply charged ions is to deliver particles or droplets of the matrix/analyte mixture to an ionization zone which is simply a heated inlet to the vacuum of the mass spectrometer. Several other methods for delivering sample are demonstrated to produce nearly equivalent results. One example shows the use of an air gun replacing the laser and producing mass spectra of proteins by shooting pellets into a metal plate which has matrix/analyte applied to the opposite side and near the ion entrance inlet to the mass spectrometer. Multiply charged ions of proteins are produced in the absence of any electric field or laser and with only the need of a heated ion entrance capillary or skimmer. The commonality of the matrix with MALDI and the mild conditions necessary for formation of ions brings into question the mechanism of formation of multiply charged ions and the importance of matrix structure in this process.

Laserspray ionization (LSI) mass spectrometry (MS) allows, for the first time, the analysis of proteins directly from tissue using high performance atmospheric pressure ionization mass spectrometers. Several abundant and numerous lower abundant protein ions with molecular masses up to ∼20,000 Da were detected as highly charged ions from delipified mouse brain tissue mounted on a common microscope slide and coated with 2,5-dihydroxyacetophenone as matrix. The ability of LSI to produce multiply charged ions by laser ablation at atmospheric pressure allowed protein analysis at 100,000 mass resolution on an Orbitrap Exactive Fourier transform mass spectrometer. A single acquisition was sufficient to identify the myelin basic protein N-terminal fragment directly from tissue using electron transfer dissociation on a linear trap quadrupole (LTQ) Velos. The high mass resolution and mass accuracy, also obtained with a single acquisition, are useful in determining protein molecular weights and from the electron transfer dissociation data in confirming database-generated sequences. Furthermore, microscopy images of the ablated areas show matrix ablation of ∼15 μm-diameter spots in this study. The results suggest that LSI-MS at atmospheric pressure potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and soft ionization, multiple charging, improved fragmentation, and cross-section analysis common to electrospray ionization.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is noted for its ability to produce primarily singly charged ions. This is an attribute when using direct ionization for complex mixtures such as protein digests or synthetic polymers. However, the ability to produce multiply charged ions, as with electrospray ionization (ESI), has advantages such as extending the mass range on mass spectrometers with limited mass-to-charge (m/z) range and enhancing fragmentation for structural characterization. We designed and fabricated a novel field free transmission geometry atmopsheric pressure (AP) MALDI source mounted to a high-mass resolution Orbitrap Exactive mass spectrometer. We report the ability to produce at will either singly charged ions or highly charged ions using a MALDI process by simply changing the matrix or the matrix preparation conditions. Mass spectra with multiply charged ions very similar to those obtained with ESI of proteins such as cytochrome c and ubiquitin are obtained with low femtomole amounts applied to the MALDI target plate and for peptides such as angiotensin I and II with application of attomole amounts. Single scan acquisitions produce sufficient ion current even from proteins.

Perfluoroalkylated nanospheres have been prepared by reaction of fullerenes with a variety of fluoroalkyl radicals. The latter are generated by thermal or photochemical decomposition of fluoroalkyl iodides or fluorodiacyl peroxides. Up to 16 radicals add to C(60) to afford easily isolable fluoroalkylated derivatives. The monosubstituted radical adducts were detected by electron spin resonance in the early stages of the fluoroalkylation reactions. These spheroidal molecules are thermally quite stable, soluble in fluoroorganic solvents, chemically resistant to corrosive aqueous solutions, and more volatile than the parent fullerenes. Films of the sublimed material display properties typical for a perfluoroalkylated material.