We report on the fabrication and performance of a gel microfluidic chip interfaced to laser desorption/ionization (LDI) mass spectrometry with a time-of-flight mass analyzer. The chip was fabricated from poly(methylmethacrylate) with a poly(dimethyl siloxane) cover. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed in the channel of the microfluidic chip. After electrophoresis, the cover was removed and either the PDMS chip or the PMMA cover was mounted in a modified MALDI ion source for analysis. Ions were formed by irradiating the channel with 2.95 m radiation from a pulsed optical parametric oscillator (OPO), which is coincident with IR absorption by N-H and O-H stretch of the gel components. No matrix was added. The microfluidic chip design allowed a decrease in the volume of material required for analysis over conventional gel slabs, thus enabling improvement in the detection limit to a pmol level, a three orders of magnitude improvement over previous studies in which desorption was achieved from an excised section of a conventional gel. ( T he rapid expansion of the field of proteomics has created an increased demand for selective, sensitive, and high-throughput methods of analysis. Soft ionization methods, such as matrix-assisted desorption/ionization (MALDI) [1,2] and electrospray (ESI) [3,4] are suitable for achieving the goal of identification and structural determination of proteins. Mass spectrometry (MS) has become the primary analysis method in this area because of its high throughput, sensitivity, and high mass accuracy. However, the large number and broad concentration range of the proteins present in the expressed proteome of a typical organism requires that one or more fractionation or separation steps be performed on the sample before mass spectrometry analysis.One of the most commonly utilized techniques for protein separation is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [5][6][7][8][9]. The molecular weight of a protein is determined by the migration distance after electrophoretic separation together with several marker proteins of known molecular weights that bracket the protein of interest. Currently, most proteome analyses are based on the separation of complex protein mixtures by 1D or 2D gel electrophoresis, followed by mass spectrometric analysis of proteolytic digests of gel spots to identify individual proteins [10 -13]. However, this protocol requires intermediate sample processing steps such as extraction of the protein from the gel [13], blotting the protein onto a membrane [14], or electroelution [15], which introduces a number of laborious and time-consuming manual steps that are difficult to automate.Various efforts have been directed toward direct analysis of gel and planar chromatography separations by laser desorption ionization (LDI) mass spectrometry. The mass spectrometric analysis of peptides and proteins by UV-MALDI directly from polyacrylamide gels has been reported previously [16]. Here, ultrathin gels (less than 10 m) ...