Laserspray Ionization, a New Atmospheric Pressure MALDI Method for Producing Highly Charged Gas-phase Ions of Peptides and Proteins Directly from Solid Solutions

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.Two primary differences between ESI and MALDI methods are the sample environment (solution versus solid) and the observable charge state(s) (multiply versus singly charged). The multiply charged ions observed in ESI mass spectrometry (MS) enhance the yields of fragment ions, a key benefit in structure characterization, and allow analysis of high molecular weight compounds on mass spectrometers with a limited mass-to-charge (m/z) range. In contrast, MALDI MS is ideal for the analysis of heterogeneous samples because it often requires less sample, and spectra of singly charged ions are easier to interpret. We report here the astonishing observation of highly charged molecular ions by laser ablation of a solid matrix/analyte mixture typically used in MALDI MS analyses. The distribution and abundances of the observed ions are similar to those obtained by ESI. Importantly, the MALDI mechanism that produces singly charged ions can be “turned on” at the operator''s will by changing only the matrix or matrix preparation conditions; this capability is not available with any other ionization method. These findings show for the first time that singly charged ions as well as multiply charged ions are available in MALDI. Besides having important mechanistic implications relating to MALDI and ESI, our findings have enormous practical analytical utility.ESI and MALDI combined with MS revolutionized the study of biological materials and earned the Nobel Prize in Chemistry for their ability to ionize proteins for analysis using MS. However, after two decades of extensive studies, the mechanism for ion formation in MALDI remains controversial (1–8). At the heart of these debates lies the predominance of singly charged ions in MALDI mass spectra; the exception being very high mass compounds. A mechanism for the formation of multiply charged ions in MALDI has previously been proposed (1) based on molecular modeling studies (9, 10) and glimpses of multiply charged ions have been observed in lower molecular weight compounds (11–14). The formation of these multiply charged ions has been attributed to sample preparation, high laser fluence, a metal-free sample stage, use of an IR laser, and atmospheric pressure (AP)1 conditions. Multiply charged ions were also recently observed by laser ablation of a liquid surface in the presence of a high electric field (15). The inability in that experiment to observe ions from a solid MALDI matrix/analyte sample or in the absence of an electric field suggests an ionization process involving liquid droplets in a high field similar to ESI (16) or other liquid based, field-induced ionization methods (17, 18).Here, we show analytically useful ESI-like MALDI mass spectra obtained using standard MALDI conditions but using a nontraditional source (19) mounted in place of the standard atmospheric pressure ionization source on a mass spectrometer most commonly used with ESI. The utility of this MALDI MS method for extending the mass range of mass spectrometers as well as the capability of peptide/protein sequencing using electron transfer dissociation (ETD) (20) is demonstrated. Because highly charged ions have not previously been observed with any MALDI ion source configuration, we briefly discuss the fundamental concepts that lead to their production. Key aspects of laserspray ionization (LSI) are laser ablation using a UV laser aligned in transmission geometry (TG) (21–23), field-free (FF) at AP (24), using a heated AP to vacuum ion transfer capillary. In order to emphasize the MALDI sample preparation but distinguish laserspray from conventional AP-MALDI, the new ionization method will hereafter be referred to as FF-TG AP-MALDI.