Automated Proteomics of E. coli via Top-Down Electron-Transfer Dissociation Mass Spectrometry Maureen K. Bunger, Benjamin J. Cargile, Anne Ngunjiri, Jonathan L. Bundy,* and James L. Stephenson, Jr. Mass Spectrometry Research Program, Research Triangle Institute, 3040 Cornwallis Road, Research Triangle Park, North Carolina 27709 Electron-transfer dissociation (ETD) has recently been introduced as a fragmentation method for peptide and protein analysis. Unlike collisionally induced dissociation (CID), fragmentation by ETD occurs randomly along the peptide backbone. With the use of the sequences deter- mined from the protein termini and the parent protein mass, intact proteins can be unambiguously identified. Because of the fast kinetics of these reactions, top-down proteomics can be performed using ETD in a linear ion trap mass spectrometer on a chromatographic time scale. Here we demonstrate the utility of ETD in high-throughput top-down proteomics using soluble extracts of E. coli. Development of a multidimensional fractionation plat- form, as well as a custom algorithm and scoring scheme specifically designed for this type of data, is described. The analysis resulted in the robust identification of 322 different protein forms representing 174 proteins, com- prising one of the most comprehensive data sets as- sembled on intact proteins to date. One of the greatest challenges currently being addressed by the discipline of analytical chemistry is the efficient analysis of proteomes, i.e., the entire protein complement of an organism. Mass spectrometry (MS) has been established as the technique of choice for large-scale protein analysis and identification. 1-3 For much proteomics work, this is accomplished two-dimensional gel electrophoresis (2-DE), followed by in-gel digestion of the sepa- rated proteins and mass spectrometric analysis. Because this analysis provides information concerning protein isoelectric point and relative molecular weight along with mass and sequence of the proteolytic peptides, one can trace not only protein expression but also protein modifications that may result in response to stimuli. Although 2-D gels are often the platform of choice for proteome-scale differential display analysis, the recovery of digested peptides from gels is relatively inefficient and can introduce additional modifications such as methionine oxidation, acrylamide adduction, and methylation of aspartic acid. In addition, for the aforementioned reasons, although the migration pattern on a 2-D gel may reveal post-translational modification (PTM) status, determining the identity of the PTM via in-gel digestion is often difficult or impossible. 4 In the past decade, much effort has been directed toward developing the use of liquid chromatography followed by tandem mass spectrometry (MS/MS) technology to analyze proteolyic digests of an entire complex sample, in what has come to be known as shotgun proteomics. The use of multidimensional separation techniques such as strong cation exchange and iso- electric focusing, followed by reversed-phase chromatography for separating complex peptide mixtures in conjunction with MS ana- lysis has facilitated the almost routine identification of thousands of proteins 5-10 from a single sample in 24-48 h of analysis time. Although the advent of shotgun proteomics has allowed large- scale robust identifications of proteins from complex samples, the capacity of this technique to determine the full complement of a proteome, including post-translational modifications, is limited. This has led to development in parallel of “top-down” proteomics technology. This approach, pioneered by McLafferty and co- workers, was made possible by the integration of electrospray ionization with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry technology. These systems have the capacity to obtain accurate mass measurements and routinely resolve charge states of highly charged ions. 11 Initial efforts focused on using accurate mass measurement and MS/MS analysis of intact protein ions as a means of protein identification. 12-14 Further developments, including integration of quadrupolar ion accumula- tion 15 and the development of hybrid ion trapping-FT-MS instru- ments, 16 have made this type of experiment possible on a chromatographic time scale and enabled the rapid identification * To whom correspondence should be addressed. E-mail: bundyj@rti.org. (1) Gygi, S. P.; Aebersold, R. Curr. Opin. Chem. Biol. 2000, 4, 489-494. (2) Godovac-Zimmermann, J.; Brown, L. R. Mass Spectrom. Rev. 2001, 20,1-57. (3) Chalmers, M. J.; Gaskell, S. J. Curr. Opin. Biotechnol. 2000, 11, 384-390. (4) Gilar, M.; Bouvier, E. S. P.; Compton, B. J. J. Chromatogr., A 2001, 909, 111-135. (5) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682. (6) Wolters, D. A.; Washburn, M. P.; Yates, J. R., III. Anal. Chem. 2001, 73, 5683-5690. (7) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-247. (8) Cargile, B. J.; Bundy, J. L.; Freeman, T. W.; Stephenson, J. L., Jr. J. Proteome Res. 2004, 3, 112-119. (9) Cargile, B. J.; Sevinsky, J. R.; Essader, A. S.; Stephenson, J. L., Jr.; Bundy, J. L. J. Biomol. Tech. 2005, 16, 181-189. (10) Cargile, B. J.; Talley, D. L.; Stephenson, J. L., Jr. Electrophoresis 2004, 25, 936-945. (11) Henry, K. D.; Williams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9075-9078. (12) Loo, J. A.; Quinn, J. P.; Ryu, S. I.; Henry, K. D.; Senko, M. W.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 286-289. (13) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 2801- 2808. (14) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806-812. Anal. Chem. 2008, 80, 1459-1467 10.1021/ac7018409 CCC: $40.75 © 2008 American Chemical Society Analytical Chemistry, Vol. 80, No. 5, March 1, 2008 1459 Published on Web 01/30/2008