Protein incorporation into MALDI-matrix crystals investigated by high resolution field emission scanning electron microscopy

Abstract

In this study, high resolution field emission scanning electron microscopy (FE-SEM) was tested for its feasibility to investigate analyte incorporation into and analyte distribution in slowly grown crystals of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2,6-dihydroxybenzoic acid (2,6-DHB); both compounds function as a matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). To investigate matrix–analyte interaction in 2,5-DHB crystals two Au-labels (20 nm colloidal gold and Nanogold) were examined as potential protein markers to visualize proteins in matrix solids by SEM. For this purpose, analyte-doped 2,5-DHB crystals were mechanically cleaved and SEM-micrographs of the opened inner faces were recorded. During the course of the Au-label study, crystal defects became apparent for crystals grown from analyte-free and analyte-doped matrix solutions; these defects are interpreted as ‘fluid’ or ‘liquid inclusions.’ The size and amount of liquid inclusions depend on the individual cooling rate during crystal growth and on the size of analyte molecules added to the matrix solution prior to crystal growth. Considering the results obtained for both matrix compounds, we assume that analyte incorporation occurs via a ‘solid solution’ in the case of 2,5-DHB as earlier proposed and not via phase defects such as liquid inclusions. Whether liquid inclusions—presumably filled with saturated mother solution—help to facilitate the desorption/ionization event remains open.