Laserspray ionization, a new atmospheric pressure MALDI method for producing highly charged gas-phase ions of peptides and proteins directly from solid solutions

Abstract

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.

Fig. 1.

MALDI (A) and ESI (B) mass spectra of a 1 pmol/μl solution of lysozyme (1-s acquisitions, 100,000 resolution (m/Δm, full-width half-height at m/z 200)), charge states 8+ to 13+ for FF-TG AP-MALDI (1 pmol in 2,5-DHB loaded on a glass microscope slide) (A) and 8+ to 11+ for ESI (1 pmol injected into 1:1 ACN/water infused at 2 μl min⁻¹) (B). Insets show the isotope distributions for the most abundant peaks and charge 10+ in each spectrum. The molecular weight can be calculated from each m/z value by knowledge of z, which is readily determined from the m/z spacing between ¹³C isotope peaks.

Scheme 1.

Schematic representation of the FF-TG AP-MALDI source and active ionization mechanisms. A, representation of the cluster model in which the absorption of photons by the matrix results in a free jet expansion, producing highly charged matrix/analyte clusters that become desolvated in the ion transfer tube, producing multiply charged ions. Ionization occurs over distances measured in mm. B, a representation of an expansion of A showing the chemical ionization process that occurs primarily near the sample surface and leads to primarily singly charged ions. This ionization process occurs in the first few μm from the surface.

Fig. 2.

Ion count versus ion transfer tube temperature plot for the 1+ through 3+ charge states of angiotensin I. The 2+ and 3+ ions are produced by the solvent-based and the 1+ ions are produced by the solvent-free MALDI sample preparation methods. The plot suggests two distinctly different ionization mechanisms, one of which (multiply charged ion formation) is highly dependent on the ion transfer tube temperature.

Fig. 3.

The FF-TG AP-MALDI mass spectrum obtained at 100,000 mass resolution from 40 fmol of bovine pancreas insulin loaded on the glass slide MALDI target plate in 2,5-DHB using the solvent-based dried droplet method. The insets show the ¹³C isotopic distribution of the 4+ and 5+ charge state ions. Rel. Abund., relative abundance.

Fig. 4.

A, the FF-TG AP-MALDI mass spectrum of a single scan acquisition from ∼5 pmol of ubiquitin in 2,5-DHB loaded onto the glass slide. B, the single scan ETD acquisition from the mass-selected 11+ charge state (m/z 779) ions. C, the summed (40-s acquisition) proton transfer spectrum of the m/z 779 ion with the sequence coverage shown.