Analyte distributions in MALDI samples using MALDI imaging mass spectrometry

The analyte distributions in matrix-assisted laser desorption/ionization (MALDI) samples have been studied using MALDI imaging at better than 10 μm spatial resolution in an orthogonal-injection TOF instrument. The technique is demonstrated by mapping the analyte distribution in typical preparations of MALDI samples using the common matrices 2,5-dihydroxybenzonic acid (2,5-DHB), sinapinic acid and 4-hydroxy-α-cyanocinnamic acid (α-HCCA). These results show evidence of exclusion of impurities, and confirm that smaller matrix crystal size gives better reproducibility from spot to spot. Large single crystals of DHB and sinapinic acid were grown to examine the incorporation of analytes within the crystals. Purified protein analytes were found to be homogenously incorporated in both types of crystal, with no evidence for preferred crystal faces. The distributions of analytes in simple mixtures in single crystals of DHB were also examined. Segregation of some species was observed and appeared to correlate with analyte hydrophobicity, and to a lesser extent analyte mass or mobility. Similar segregation phenomena were observed with fluorescence microscopy of analytes labeled with fluorescent dyes in large 2,5-DHB single crystals, and in smaller crystals grown by fast evaporation. The above investigations may shed some light on optimizing sample preparation with different matrices.