Chromatographic Separation and Sample Preparation in One Step for MALDI Mass Spectrometric Analysis of Subpicomole Amounts of Heterogeneous Protein Samples

摘要

A rapid and efficient sample preparation method is described that allows analysis of biopolymer mixtures by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Very low amounts of complex biopolymer mixtures can be analyzed by MALDI MS without any apparent loss of material and with a substantial gain in time by combining chromatographic separation and sample preparation in a single operation. This micropurification method is based on both the formation of a submicroliter reversed-phase chromatographic bed and the use of matrix-containing eluent solutions. In this way, desalting, matrix mixing, and micropurification of a biological Sample could be performed in one step. A proteolytic mixture containing Ca↑(2+) ATPase fragments ranging from ~1000 to ~30 000 Da, obtained after endoproteinase AspN cleavage of sarcoplasmic reticulum vesicles, was analyzed by MALDI MS. Direct analysis by MALDI MS of this peptidic mixture did not provide any signal for the higher molecular mass species. When our micropurification technique was applied to this sample, successful mass data acquisitions for as low as 150 fmol of the ~30 000-Da fragment were performed.