Nanoflow gradient generator coupled with mu-LC-ESI- MS/MS for protein identification

The large-scale identification of proteins from proteomes of complex organi sms, and the availability of various types of protein and DNA databases, in creasingly require the additional information provided by tandem mass spect rometry. HPLC and mu LC coupled to ESI-MS/MS presently dominate the field o f protein identification by tandem mass spectrometry and database searching , The analysis of protein digests is typically performed using HPLC or LC c olumns with 50-100-mum diameters, requiring the delivery of solvent gradien ts at low to mid nanoliter per minute flow rates. This has been typically a chieved using expensive generic HPLC pumping systems for the delivery of mi croliter per minute Gradients that were either flow-split or sampled. Here we present an alternative system for the delivery of nanoliter per minute g radients. The inexpensive nanoflow gradient generator (eta grad) described here can be modulated to reproducibly deliver selected gradients. The perfo rmance of the eta grad on-line with a mu LC-ESI-MS/MS system has been demon strated for the identification of standard protein digests. Moreover, the p erformance of the eta grad-mu LC-ESI-MS/MS system, with protein prefraction ation by IPG isoelectric focusing, was also evaluated for rapid study of ye ast and human proteomes.