Direct Acquisition of Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectra from Laser Capture Microdissected Tissues

Abstract

Recent studies have demonstrated the usefulness of laser capture microdissection (LCM) in gene expression studies of pure cell populations derived from healthy and diseased tissues (1)(2)(3). However, mRNA expression does not necessarily correlate with protein abundance or predict posttranslational modifications (4). Currently, a few groups have reported details of protein expression profiling of cells captured by LCM, using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by peptide mass fingerprinting of selected proteins (5)(6). Although such an approach represents the gold standard for global analysis of protein expression, 2D-PAGE often is impractical for diagnostic applications. We describe a rapid and sensitive method to obtain an abridged protein expression profile from microdissected cells by the direct acquisition of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectra from LCM transfer films. LCM uses an infrared laser to transiently and focally melt an ethylene vinyl acetate (EVA) thermoplastic film applied to a tissue section. During the infrared laser pulse, the EVA transfer film fuses with the underlying cells, which remain adherent when the film is separated from the tissue section. In most applications, the transfer film is subsequently immersed in a lysis buffer to extract soluble molecules from the captured cells and their microenvironment, and the lysate is used in further analysis. Our approach is to obtain MALDI-ToF mass spectra directly from microdissected cells of interest. MALDI-ToF allows the simultaneous molecular mass characterization of a broad variety of biological molecules up to 100 kDa or higher. Mass measurements of 1 pmol of an analyte present in a 1-μL specimen are routine for pure analytes and simple mixtures. …