Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry

A simple reversed-phase nano-column purification and sample preparation tec hnique is described, which markedly improves the mass spectrometric analysi s of complex and contaminated peptide mixtures by matrix-assisted laser des orption/ionization (MALDI). The method is simple, fast and utilizes only lo w-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 so lution 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 s ample spots. This simplifies the analysis and minimizes discrimination effe cts due to sample heterogeneity, because the desorption/ionization laser si multaneously irradiates a large portion of the sample. Taken together, thes e features of the method significantly improve the sensitivity for MALDI/MS analysis of contaminated peptide samples compared with the commonly used s ample preparation procedures. This is demonstrated with in-gel tryptic dige sts of proteins from human brain that were separated by 2D gel electrophore sis. Furthermore, it is shown that with this method 2,5-dihydroxybenzoic ac id (DHB) acts as an efficient matrix for peptide mapping. Both detection se nsitivity and sequence coverage are comparable to those obtained with the c urrently preferred matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The hi gher stability of peptide ions generated with DHB compared with CHCA is adv antageous when analyzing fragile sample molecules. Therefore, the method de scribed here is also of interest for the use of Fourier transform ion cyclo tron resonance (FT-ICR) or ion-trap mass analyzers. Copyright (C) 1999 John Wiley & Sons, Ltd.