Generation of multiply charged ions from homogeneous MALDI microcrystals

Abstract

The homogeneity of matrix assisted laser desorption/ionization (MALDI) microcrystals, laser fluence, and the proton affinity (PA) of matrices determine the generation of multiply charged ions. Here we adopt three structurally similar matrices, i.e. α-cyano-4-hydroxycinnamic acid (CHCA), 4-chloro-α-cyanocinnamic acid (Cl-CCA), and (E)-propyl α-cyano-4-hydroxyl cinnamylate (CHCA-C3) to examine the homogeneity of microcrystals by measuring the relative standard deviation (RSD) values of the matrices with the homogeneous sample preparation method, e.g. forced dried droplet (FDD). The average RSD values of CHCA, Cl-CCA, and CHCA-C3 matrices are ∼15%, ∼10%, and ∼20%, respectively. With the same CHCA matrix, we found the charge state of myoglobin ions can obtain more charges (+6) with the FDD method as compared to +5 charges obtained by the inhomogeneous sample preparation method, i.e. dried droplet (DD). The laser fluence also affects the generation of multiply charged ions. For instance, the maximum charge states of myoglobin and cytochrome c ions are +6 with CHCA and +4 with CHCA-C3 at threshold laser fluence and decreases at elevated laser fluence. The charge state of myoglobin ions with Cl-CCA matrix is +3 at threshold laser fluence and increases to the maximum charge state of +6 and eventually reduces to +5 with elevated laser fluence. Moreover, we found low PA value of matrix results in high charge state of analyte ions and vice versa. The matrix PA modulates the proton transfer reaction, leading to the formation of multiply charged ions. The homogeneous samples help control the charge states of ions and generate the multiply charged protein ions which can be used as potential mass calibration standards to search suitable protein samples at high m/z ratios with a MALDI time-of-flight mass spectrometer.