Two-layer sample preparation: A method for MALDI-MS analysis of complex peptide and protein mixtures

The analytical performance of matrix-assisted laser desorption/ionization ( MALDI) mass spectrometry for direct analysis of peptide and protein mixture s is strongly dependent on the sample and matrix preparation. A two-layer s ample preparation method is demonstrated to be very effective for analyzing complex mixtures. In this method, the first layer on the MALDI probe is th e densely packed matrix microcrystals formed by fast solvent evaporation of a matrix solution. A mixture solution containing both matrix and sample is then deposited onto the first layer to form uniform analyte/matrix microco crystals. It is found that the addition of matrix to the second-layer sampl e solution proves to be critical in analyzing mixtures of peptides and prot eins covering a broad mass range. The effect of solvent conditions for prep aring the second-layer solution is discussed. The application of this metho d is demonstrated for the analysis of cow's milk where milk proteins as wel l as peptide fragments produced from proteins by indigenous proteinases are detected. Direct analyses of peptides and proteins from a bacteria extract and crude egg white are also illustrated.