A simple reversed-phase nano-column purification and sample preparation technique is described, which markedly improves the mass spectrometric analysis of complex and contaminated peptide mixtures by matrix-assisted laser desorption/ionization (MALDI). The method is simple, fast and utilizes only low-cost disposables. After loading the sample on the column and a subsequent washing step, the analyte molecules are eluted with 50-100 nl of matrix solution directly on to the MALDI/MS target. The washing step ensures removal of a wide range of contaminants. The small bed volume of the column allows efficient sample concentration and the elution process yields very small sample spots. This simplifies the analysis and minimizes discrimination effects due to sample heterogeneity, because the desorption/ionization laser simultaneously irradiates a large portion of the sample. Taken together, these features of the method significantly improve the sensitivity for MALDI/MS analysis of contaminated peptide samples compared with the commonly used sample preparation procedures. This is demonstrated with in-gel tryptic digests of proteins from human brain that were separated by 2D gel electrophoresis. Furthermore, it is shown that with this method 2,5-dihydroxybenzoic acid (DHB) acts as an efficient matrix for peptide mapping. Both detection sensitivity and sequence coverage are comparable to those obtained with the currently preferred matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The higher stability of peptide ions generated with DHB compared with CHCA is advantageous when analyzing fragile sample molecules. Therefore, the method described here is also of interest for the use of Fourier transform ion cyclotron resonance (FT-ICR) or ion-trap mass analyzers.