Anal. Chem. 2001, 73, 1307-1315
Nanoflow Gradient Generator Coupled with µ-LC-ESI-MS/MS for Protein Identification Thierry Le Bihan,†,‡ Devanon Pinto,‡,§ and Daniel Figeys*,‡
Kinetek Pharmaceuticals, Inc., 1779 West 75th Avenue, Vancouver, BC, Canada, MDS-Sciex, 71 Four Valley Drive, Concord, ON, Canada, and Institute for Marine Biosciences, National Research CouncilsCanada, 1411 Oxford Street, Halifax, NS, Canada
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 flowsplit 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. The emergence of proteomics as an enabling postgenome technology is requiring the rapid development of new analytical techniques to satisfy the ever-increasing need for ultrahigh sensitivity and rapid protein identification. Mass spectrometry, based either on MALDI-TOF1-3 or ESI-MS/MS,3-5 is rapidly dominating the field. Furthermore, it is apparent that, with the completion of large DNA sequencing projects and the increasing * Corresponding author. Present address: MDS-Ocata, 480 University Ave., Suite 401, 4th floor, Toronto, ON, Canada, M5G1V2. (e-mail)
[email protected]. † Kinetek Pharmaceuticals, Inc. ‡ National Research CouncilsCanada. § MDS-Sciex. (1) Li, G.; Waltham, M.; Anderson, N. L.; Unsworth, E.; Treston, A.; Weinstein, J. N. Electrophoresis 1997, 18, 3-4. (2) Patterson, S. D.; Thomas, D.; Bradshaw, R. A. Electrophoresis 1996, 17, 877-891. (3) Patterson, S. D.; Aebersold, R. In Protein Analysis and Design; HogueAngeletti, R., Ed.; Academic Press: New York, 1998; pp 3-120. (4) James, P. Biochem. Biophys. Res. Commun. 1997, 231, 1-6. (5) Wilkins, M. R.; Pasquali, C.; Appel, R. D.; Ou, K.; Golaz, O.; Sanchez, J. C.; Yan, J. X.; Gooley, A. A.; Hughes, G.; HumpherySmith, I.; Williams, K. L.; Hochstrasser, D. F. Bio Technology 1996, 14, 61-65. 10.1021/ac000948b CCC: $20.00 Published on Web 02/16/2001
© 2001 American Chemical Society
availability of express sequence tag (EST)6,7 and single-nucleotide polymorphism (SNP)8,9 databases, tandem mass spectrometry (MS/MS) for the identification of proteins is becoming even more essential to large-scale proteomic platforms. Currently, MS/MS analysis of protein digests is typically achieved using ESI-based mass spectrometers, requiring suitable sample introduction systems to the electrospray interface. Already a small but growing suite of technologies based on continuous infusion or separation techniques is becoming available.10-17 Among these techniques, liquid chromatography coupled with ESI-MS/MS2,15,18-20 has proved to be the most robust and userfriendly technique for the rapid analysis and identification of proteins. Furthermore, the implementation of small-bore LC columns (
