A novel protocol of rapid and automatic on-plate desalting (OPD) and peptide concentration for 2-DE-MALDI-MS has been developed by the approach of templating the hydrophobic polymer solution over Kapton-etched mask. For the template technique, small hydrophobic polymer [linear poly(methyl methacrylate) (PMMA), PMMA derivatized with fullerene-C60 (PMMA-C60), linear polystyrene (PSt), or PSt derivatized with fullerene-C60 (PSt-C60)] spots (990 microm od) are patterned at the centers of stainless MALDI plate wells (1400 microm id). Tryptic-peptide solution with no predesalting was dropped onto the central hydrophobic spots, resulting in a concentration of proteolytic peptides on the hydrophobic polymer surface with a reduced spot size. The dried peptide layer was then covered subsequently with over-volume matrix solution, causing the removal of redissolved salts from the spot center to the spot edge by means of a natural "outward flow." The proposed OPD protocol exhibited a dramatic enhancement in S/N up to 850 for 14 fmol BSA digests in the coexistence of 100 mM salts, compared with barely detectable peaks in ordinary way. This analysis has shown that the success rate of identification was increased by two-fold for low abundance proteins in the human liver tissue with no need for the conventional ZipPlate desalting strategy.