Degree of Ionization in MALDI of Peptides: Thermal Explanation for the Gas-Phase Ion Formation

Abstract

Degree of ionization (DI) in matrix-assisted laser desorption ionization (MALDI) was measured for five peptides using α-cyano-4-hydroxycinnanmic acid (CHCA) as the matrix. DIs were low 10(-4) for peptides and 10(-7) for CHCA. Total number of ions (i.e., peptide plus matrix) was the same regardless of peptides and their concentration, setting the number of gas-phase ions generated from a pure matrix as the upper limit to that of peptide ions. Positively charged cluster ions were too weak to support the ion formation via such ions. The total number of gas-phase ions generated by MALDI, and that from pure CHCA, was unaffected by the laser pulse energy, invalidating laser-induced ionization of matrix molecules as the mechanism for the primary ion formation. Instead, the excitation of matrix by laser is simply a way of supplying thermal energy to the sample. Accepting strong Coulomb attraction felt by cations in a solid sample, we propose three hypotheses for gas-phase peptide ion formation. In Hypothesis 1, they originate from the dielectrically screened peptide ions in the sample. In Hypothesis 2, the preformed peptide ions are released as part of neutral ion pairs, which generate gas-phase peptide ions via reaction with matrix-derived cations. In Hypothesis 3, neutral peptides released by ablation get protonated via reaction with matrix-derived cations.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: Mechanistic Studies and Methods for Improving the Structural Identification of Carbohydrates

“…[27][28][29][30][31][32] For analytes with lower proton a nity, such as carbohydrates, the ionization e ciency drops by a few orders of magnitude to 10 −7 -10 −8 . 23,33,34) A better understanding of mechanisms leads to improvements in the ionization e ciency and increases its e ectiveness toward more elds. Over the last three decades, fundamental ionization mechanisms have been systematically studied by manipulating one or more parameters among di erent matrix systems.…”

Efficiency of Gas-Phase Ion Formation in Matrix-Assisted Laser Desorption Ionization with 2,5-Dihydroxybenzoic Acid as Matrix

“…4,5 In contrast, in the second group, photo-excitation is simply an instantaneous way of supplying thermal energy to the sample and hence inducing its ablation. [6][7][8][9] The thermal energy needed for ablation is supplied by photo-excitation in the first group of hypotheses also. Ionization of matrix molecules via pooling of excitons is the leading hypothesis belonging to the first group, while thermal processes such as the pre-formed ion emission have been proposed for the second group.…”

“…4 Recently, we measured the number of gas-phase ions generated in MALDI of peptides using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. 9 Two important observations were made in the study. One was that the total number of gas-phase ions appearing in a MALDI spectrum, viz.…”

“…As can be seen from the same table, the total number of ions appearing in MALDI spectra, i.e., the sum of the numbers of matrix-and analyte-derived ions, is the same regardless of the analyte concentration in solid sample. Hence, as in CHCA-MALDI, 9 [M + H] + is the primary ion and the matrixto-peptide proton transfer generates [P + H] + . This conclusion seems to be valid even though the analyte-to-peptide alkali metal ion transfer also occurs in the present case.…”

“…The ionization efficiency for pure DHB was around one fifth of that for CHCA, (2.4 ± 1.2) × 10 −8 for DHB vs. (1.2 ± 0.1) × 10 −7 for CHCA. 9 When compared at comparable concentrations, the ionization efficiency for Y 5 R in DHB-MALDI was smaller than that in CHCA-MALDI by a factor of 3-4, for example, (5.5 ± 0.8) × 10 estimated from the data reported by Sundqvist et al 21 We would like to emphasize that both of them are smaller by orders of magnitude than 10 , is generated by a some form of laser-induced ionization such as the multi-photon ionization mediated by exciton pooling, the efficiency for its formation will increase with the photon energy density of the irradiated spot. The energy density will certainly increase with the laser pulse energy.…”