Single-step perfusion chromatography with a throughput potential for enhanced peptide detection by matrix-assisted laser desorption/ ionization-mass spectrometry.

Mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis can be routinely performed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) which has become a standard tool. Since MALDI-MS detection relies heavily on the quality of the MALDI target, development of an efficient sample preparation technique for removal of sample contaminants is necessary. To date, among the several sample preparation techniques for MALDI targets available, multistep perfusion chromatography (MSPC) using Poros R2 and Oligo R3 has been most commonly used. However, MSPC requires at least four working steps and is not efficient for high-throughput analysis and recovery of low abundance proteins. During the course of proteomic analysis of a large set of rat liver tissues and the immortalized human sebaceous gland cells (SZ95 cells), we were interested in developing an alternative to MSPC. Here, we describe a single-step perfusion chromatography (SSPC) method for MALDI target preparation, which uses a tiny column packed with a mixture of Poros R2 and Oligo R3 resins. The SSPC method significantly improves not only detection of peptides but also efficiency of sample handling, thus enabling high-throughput sample preparation for analyzing large set of samples with high resolution and reproducibility.