Nanoflow Gradient Generator Coupled with μ-LC−ESI-MS/MS for Protein Identification

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Abstract
The large-scale identification of proteins from proteomes of complex organisms, and the availability of various types of protein and DNA databases, increasingly require the additional information provided by tandem mass spectrometry. HPLC and μLC coupled to ESI-MS/MS presently dominate the field of protein identification by tandem mass spectrometry and database searching. The analysis of protein digests is typically performed using HPLC or LC columns with 50−100-μm diameters, requiring the delivery of solvent gradients at low to mid nanoliter per minute flow rates. This has been typically achieved using expensive generic HPLC pumping systems for the delivery of microliter per minute gradients that were either flow-split or sampled. Here we present an alternative system for the delivery of nanoliter per minute gradients. The inexpensive nanoflow gradient generator (ηgrad) described here can be modulated to reproducibly deliver selected gradients. The performance of the ηgrad on-line with a μLC−ESI-MS/MS system has been demonstrated for the identification of standard protein digests. Moreover, the performance of the ηgrad-μLC−ESI-MS/MS system, with protein prefractionation by IPG isoelectric focusing, was also evaluated for rapid study of yeast and human proteomes.