Factors that affect the multiply charge formation of protein ions in a MALDI process

Avinash Patil^1*, Ly Khánh¹, Cheng-Kang Chiang², Wen-Ping Peng¹

¹Physics, National Dong Hwa University, Hualien, Taiwan
²Chemistry, National Dong Hwa University, Hualien, Taiwan

* Presenter:Avinash Patil, email:avi.smash4@gms.ndhu.edu.tw

Over the past three decades, matrix-assisted laser desorption/ionization (MALDI) has been widely adopted for the analysis of nonvolatile and fragile molecules, especially peptides and large proteins due to the low charge state distribution of ions. However, multiply charged MADLI ion generation has been not studied exclusively. In this report we have examined the effect of the photophysical and chemical properties of matrix molecules on the generation of multiply charged protein ions. Three matrices i.e. α-cyano-4-hydroxyl cinnamylate (CHCA), 4-chloro-α-cyanocinnamic acid (Cl-CCA), and (E)-propyl α-cyano-4-hydroxyl cinnamylate (CHCA-C3) having similar chemical structure with different proton affinity (PA) were examined to form the multiply charged ions with homogeneous, i.e. forced dried droplet (FDD) and heterogeneous i.e. dried droplet (DD) sample preparation methods. Myoglobin (Myo) sample prepared by FDD method can form highly homogeneous sample surface with Cl-CCA matrix and the relative standard deviation (RSD) is ~ 10% which was a factor of two better than the DD method (RSD ~ 45%). The RSD values of CHCA and CHCA-C3 are ~ 16% and ~ 20%. Homogeneous sample surface can generate reproducible and higher charge states (~ +6 charge state) of Myo ions than the inhomogeneous sample surface (< +4 charge state). In addition, laser fluence affects the formation of multiply charged ions. The maximum charge states of analytes were obtained at threshold laser fluence, i.e. minimum laser fluence, for CHCA and CHCA-C3 matrices and at moderate laser fluence for Cl-CCA matrix. The ion signal intensity of lower charge state ions increased rapidly with elevated laser fluence than that of the higher charge state ions and eventually the higher charge state ions disappeared at higher laser fluence conditions. Moreover, higher PA value (e.g. CHCA-C3) tends to obtain lower charge states (+4 charge states) of analyte ions and vice versa (e.g. low PA Cl-CCA matrix gets +6 charge states. Thereby, gas phase protonation plays a significant role for secondary ionization in a MALDI plume and the PA values of matrices affect the proton transfer reaction in a gaseous plume and determine the charge states of analyte ions. We found the generation of multiply charged ions is not only an intrinsic property of matrix molecules defined by the PA of matrix, but the formation of homogeneous MALDI microcrystals and the selection of optimized laser fluence are also crucial. The multiply charged protein ions can be adopted for mass calibration at the high mass ranges with MALDI TOF-MS.

Keywords: Multiply charged MALDI ions, Laser fluence, Homogeneous sample surface, Proton affinity, MALDI-TOF mass spectrometer