High-Throughput Identification of In-Gel Digested Proteins by Rapid, Isocratic HPLC/MS/MS

The high sensitivity and accuracy of mass spectrometry for identifying proteins has led to an explosive expansion of proteomics research, necessitating rapid procedures for HPLC/MS/MS analysis. Current HPLC/MS/MS analysis usually relies on elution of peptides from the HPLC column with a gradient that takes a total of 45-70 min for each cycle, limiting sample throughput and the speed of protein identification. Here we report a simple method for high-throughput protein identification, using isocratic, either methanol- or acetonitrile-based buffer systems, HPLC elution into an LTQ mass spectrometer. This procedure allows each cycle of highly sensitive