To have a clearer understanding on the ionization process in matrix assisted laser desorption/ionization (MALDI), we introduced a new 'analyte-support' mechanism. Using the mixed crystal of 2,4,6-trihydroxyacetophenone (THAP) and analyte molecules such as Angiotensin II (AngII), crystal shape changing was observed and a new band appeared in diffuse reflectance spectrum. Furthermore, it was observed that the peak ratio of [analyte + H](+)/[THAP + H](+) in MALDI mass spectra changed simultaneously with the change of in the mixing ratio of analytes. The experimental results have provided direct evidence that interaction among the polar analyte, matrix and water molecules are important for the process of ionization. (C) 2014 Elsevier B.V. All rights reserved.