A Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Oct 30, 2004 - Helen J. Cooper,*,† John K. Heath,† Ellis Jaffray,‡ Ronald T. Hay,‡ TuKiet T. ... University of St. Andrews, St. Andrews, Scotl...
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Anal. Chem. 2004, 76, 6982-6988

Identification of Sites of Ubiquitination in Proteins: A Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Approach Helen J. Cooper,*,† John K. Heath,† Ellis Jaffray,‡ Ronald T. Hay,‡ TuKiet T. Lam,§ and Alan G. Marshall§,|

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K., Centre for Biomolecular Sciences, University of St. Andrews, St. Andrews, Scotland, Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, and Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32310

Structural elucidation of posttranslationally modified peptides and proteins is of key importance in the understanding of an array of biological processes. Ubiquitination is a reversible modification that regulates many cellular functions. Consequences of ubiquitination depend on whether a single ubiquitin or polyubiquitin chain is added to the tagged protein. The lysine residue through which the polyubiquitin chain is formed is also critical for biological activity. Robust methods are therefore required to identify sites of ubiquitination modification, both in the target protein and in ubiquitin. Here, we demonstrate the suitability of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, in conjunction with activated ion electron capture dissociation (AI ECD) or infrared multiphoton dissociation (IRMPD), for the analysis of ubiquitinated proteins. Polyubiquitinated substrate protein GST-Ubc5 was generated in vitro. Tryptic digests of polyubiquitinated species contain modified peptides in which the ubiquitin C-terminal Gly-Gly residues are retained on the modified lysine residues. Direct infusion microelectrospray FT-ICR of the digest and comparison with an in silico digest enables identification of modified peptides and therefore sites of ubiquitination. Fifteen sites of ubiquitination were identified in GST-Ubc5 and four sites in ubiquitin. Assignments were confirmed by AI ECD or IRMPD. The Gly-Gly modification is stable and both tandem mass spectrometric techniques are suitable, providing extensive sequence coverage and retention of the modification on backbone fragments.

protein in many ways, an important function of ubiquitin is to target proteins for degradation by the proteasome. In addition to degrading damaged, misfolded, and misassembled proteins, the ubiquitin-proteasome system targets proteins including transcription factors, signal transducers, tumor suppressors, and membrane receptors3,4 regulating processes such as cell cycle progression5 and inflammatory6 and immune responses.7 The first step in the modification of proteins by ubiquitin is the ATP-dependent activation of ubiquitin by the ubiquitinactivating enzyme (E1), in which a thioester bond between the side chain of a cysteine residue in E1 and the C-terminus of ubiquitin is formed. The ubiquitin is then transferred, via a transesterification reaction, to a conserved cysteine residue in an ubiquitin conjugating enzyme (E2). Finally, the ubiquitin is transferred to the target protein by formation of an isopeptide bond between the -amino group of a lysine residue in the substrate and the C-terminus of ubiquitin. Often, the final stage requires the presence of a ubiquitin ligase (E3). The ligase may act either as a ubiquitin donor or in substrate recognition.8 Following ubiquitination of a substrate, the ubiquitin may act as a nucleus for further modification. Hence, substrates become tagged with a polyubiquitin chain. While modification of substrates by the ubiquitin-like proteins SUMO and Nedd8 generally occurs at specific lysine residues,9-11 this is not usually the case for polyubiquitinated protein. Sitedirected mutagenesis of three ubiquitin substrates, c-Jun,12 the T-cell receptor ζ subunit,13 and the encephalomyocarditis virus 3C protease,14 revealed that wild-type susceptibility to ubiquitination was retained by each mutant. In contrast, NF-κB inhibitor

Ubiquitin is a highly conserved 76-residue protein found in all tissues, in all eukaryotes. The covalent conjugation of ubiquitin to other cellular proteins regulates a wide array of cell functions.1,2 Although ubiquitination may alter the properties of the modified

(3) Desterro, J. M. P.; Rodriguez, M. S.; Hay, R. T. Cell. Mol. Life Sci. 2000, 57, 1207-1219. (4) Hershko, A.; Ciechanover, A. Annu. Rev. Biochem. 1998, 67, 425-479. (5) Koepp, D. M.; Harper, J. W.; Elledge, S. J. Cell 1999, 97, 431-434. (6) Ghosh, S.; May, M. J.; Kopp, E. B. Annu. Rev. Immunol. 1998, 16, 225260. (7) Rock, K. L.; Goldberg, A. L. Annu. Rev. Immunol. 1999, 17, 739-779. (8) Hershko, A.; Ciechanover, A. Annu. Rev. Biochem. 1992, 61, 761-807. (9) Hochstrasser, M. Nat. Cell Biol. 2000, 2, E153-E157. (10) Hay, R. T. Trends Biochem. Sci. 2001, 26, 332-333. (11) Jentsch, S.; Pyrowolakis, G. Trends Cell Biol. 2000, 10, 335-342. (12) Treier, M.; Staszewski, L. M.; Bohmann, D. Cell 1994, 78, 787-798. (13) Hou, D.; Cenciarelli, C.; Jensen, J. P.; Nguyen, H. B.; Weissman, A. M. J. Biol. Chem. 1994, 269, 14244-14247.

* To whom correspondence should be addressed. Telephone: +44 (0)121 414 7527. Fax: +44 (0)121 414 5925. E-mail: [email protected]. † University of Birmingham. ‡ University of St. Andrews. § National High Magnetic Field Laboratory, Florida State University. | Department of Chemistry and Biochemistry, Florida State University. (1) Weissman, A. M. Nat. Rev. Mol. Cell. Biol. 2001, 2, 169-178. (2) Pickart, C. M. Annu. Rev. Biochem. 2001, 70, 503-533.

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IκBR is polyubiquitinated at specific lysines.15,16 Ubiquitin itself contains seven lysine residues that can serve as ubiquitination sites in the generation of polyubiquitin.17-19 Polyubiquitin chains conjugated through Lys4820 are targeted for degradation by the 26S proteasome,21 as are Lys29-linked polyubiquitin chains.22 Lys63-linked polyubiquitin regulates processes that do not involve proteolysis, e.g., ribosomal function,23 postreplicative DNA repair,24,25 the initiation of inflammatory response,26 and the function of some transcription factors.27 The roles of Lys6-, Lys11-, Lys27-, and Lys33-linked polyubiquitin are, as yet, unknown. Ligation of a single ubiquitin onto proteins has diverse consequences for their functions and can act as a signal for endocytosis28 and DNA damage responses29 and in the case of histones as a component of the “histone code”.30 Traditionally, sites of ubiquitination have been inferred from the functional consequences of introducing specific lysine mutations into target proteins (e.g., 12-14, 18, 19, 21). However, this is an indirect approach, which identifies lysine residues required for ubiquitination rather than the actual sites of modification. Recently, Gygi and co-workers developed a proteomics approach for identifying sites of ubiquitination.17 His-tagged ubiquitin conjugates were purified from whole cell lysates by nickel-affinity chromatography and digested with trypsin. The tryptic peptides were separated by strong cation exchange chromatography and fractions collected. Each fraction was then subjected to online reversed-phase liquid chromatography-tandem mass spectrometry (MS/MS). The resulting MS/MS data were searched against a protein database by use of the Sequest algorithm.31 Here, we introduce a Fourier transform ion cyclotron resonance (FT-ICR)32 mass spectrometry approach for identifying sites of ubiquitination in proteins. FT-ICR is the highest performance mass spectrometry technique in terms of resolution and mass accuracy. Consequently, FT-ICR finds many applications in the (14) Lawson, T. G.; Gronros, D. L.; Evans, P. E.; Bastien, M. C.; Michalewich, K. M.; Clark, J. K.; Edmonds, J. H.; Graber, K. H.; Werner, J. A.; Lurvey, B. A.; Cate, J. M. J. Biol. Chem. 1999, 274, 9871-9880. (15) Rodriguez, M. S.; Wright, J.; Thompson, J.; Thomas, D.; Baleux, F.; Virelizier, J. L.; Hay, R. T.; ArenzanaSeisdedos, F. Oncogene 1996, 12, 2425-2435. (16) Scherer, D. C.; Brockman, J. A.; Chen, Z. J.; Maniatis, T.; Ballard, D. W. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 11259-11263. (17) Peng, J.; Schwartz, D.; Elias, J. E.; Thoreen, C. C.; Cheng, D.; Marisischky, G.; Roelofs, J.; Finley, D.; Gygi, S. P. Nat. Biotechnol. 2003, 21, 921-926. (18) Baboshina, O. V.; Haas, A. L. J. Biol. Chem. 1996, 271, 2823-2831. (19) Wu-Baer, F.; Lagrazon, K.; Yuan, W.; Baer, R. J. Biol. Chem. 2003, 278, 34743-34746. (20) Chau, V.; Tobias, J. W.; Bachmair, A.; Marriott, D.; Ecker, D. J.; Gonda, D. K.; Varshavsky, A. Science 1989, 243, 1576-1583. (21) Gregori, L.; Poosch, M. S.; Cousins, G.; Chau, V. J. Biol. Chem. 1990, 265, 8354-8357. (22) Arnason, T.; Ellison, M. J. Mol. Cell. Biol. 1994, 14, 7876-7883. (23) Spence, J.; Gali, R. R.; Dittmar, G.; Sherman, F.; Karin, M.; D., F. Cell 2000, 102, 67-76. (24) Spence, J.; Sadis, S.; Haas, A. L.; Finley, D. Mol. Cell. Biol. 1995, 15, 12651273. (25) Hofmann, R. M.; Pickart, C. M. Cell 1999, 96, 645-653. (26) Deng, L.; Wang, C.; Spencer, E.; Yang, L.; A., B., J. X., Y., Slaughter, C., Pickart, C.; Chen, Z. J. Cell 2000, 103, 351-361. (27) Kaiser, P.; Flick, K.; Wittenberg, C.; Reed, S. I. Cell 2000, 102, 303-314. (28) Hicke, L. Trends Cell. Biol. 1999, 9, 107-112. (29) Hoege, C.; Pfander, B.; Moldovan, G. L.; Pyrowolakis, G.; Jentsch, S. Nature 2002, 419, 135-141. (30) Sun, Z. W.; Allis, C. D. Nature 2002, 418, 104-108. (31) Eng, J.; McCormack, A. L.; Yates, J. R. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989. (32) Marshall, A. G., Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35.

analysis of complex mixtures (e.g., refs 33-35). In this study, polyubiquitinated protein GST-Ubc5 was generated in vitro and subjected to trypsin digestion. As described by Peng et al.,17 modified lysine residues retain a Gly-Gly moietysthe ubiquitin C-terminussfollowing treatment with trypsin. Moreover, proteolysis at the modified lysine does not occur. The unseparated tryptic digest was analyzed by direct infusion microelectrospray (microESI)36 FT-ICR mass spectrometry. As a consequence of the mass accuracy of FT-ICR, modified peptides, and therefore sites of ubiquitination in both the substrate and ubiquitin, could be identified by mass alone. Confirmation of assignments was achieved by isolating modified peptides and subjecting them to MS/MS: either activated ion electron capture dissociation (AI ECD)37 or infrared multiphoton dissociation (IRMPD).38 The GlyGly modification is stable and both techniques are suitable for confirming identity; however, AI ECD generally resulted in improved sequence coverage. EXPERIMENTAL METHODS Preparation of Polyubiquitinated GST-Ubc5. Ubiquitin was purchased from Sigma (Poole, Dorset, U.K.) and used without further purification. Human E1 ubiquitin-activating enzyme39 was purified from recombinant baculovirus-infected insect cells by affinity chromatography on a ubiquitin matrix, as described previously.40 GST-Ubc5 was expressed in Escherichia coli and purified by glutathione agarose affinity chromatography, as described previously.41 Ubiquitin (2 mg), ubiquitin E1 (16 µg), and GST-Ubc5 (700 µg) in a final volume of 800 µL were incubated at 37 °C for 3 h in the presence of 50 mM Tris HCl pH 7.5, 5 mM MgCl2, and 2 mM ATP. Note, the GST-Ubc5 acts as both the conjugating enzyme and final substrate in this reaction. After 3 h, the reaction was terminated by the addition of EDTA to 10 mM. The reaction mixture was dialyzed against 50 mM ammonium bicarbonate (pH 8.0). Preparation of Samples for FT-ICR Mass Spectrometry. A 100-µL aliquot of the dialyzed reaction mixture was added to 250 µL of 7 µg/mL sequencing grade modified trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate, for a GST-Ubc5/ trypsin ratio of 50:1, and incubated at 37 °C overnight. A 20-µL aliquot of the tryptic mixture was desalted by use of a C18 ZipTip (Millipore, Billerica, MA) and eluted into 5 µL of 1:1 water/ acetonitrile (J. T. Baker, Philipsburg, NJ), 0.1% formic acid (Aldrich, Milwaukee, WI). A further 10 µL of 1:1 water/acetontrile, 0.1% formic was added to the eluant, and the solution was (33) Cooper, H. J.; Marshall, A. G. J. Agric. Food Chem. 2001, 49, 5710-5718. (34) Nilsson, C. L.; Cooper, H. J.; Hakansson, K.; Marshall, A. G.; Ostberg, Y.; Lavrinovicha, M.; Bergstrom, S. J. Am. Soc. Mass Spectrom. 2001, 13, 295299. (35) Qian, K.; Robbins, W. K.; Hughey, C. A.; Cooper, H. J.; Rodgers, R. P.; Marshall, A. G. Energy Fuels 2001, 15, 1505-1511. (36) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605613. (37) Horn, D. M.; Ge, Y.; McLafferty, F. W. Anal. Chem. 2000, 72, 4778-4784. (38) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (39) Rolfe, M.; Beerromero, P.; Glass, S.; Eckstein, J.; Berdo, I.; Theodoras, A.; Pagano, M.; Draetta, G. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3264-3268. (40) Rodriguez, M. S.; Desterro, J. M. P.; Lain, S.; Lane, D. P.; Hay, R. T. Mol. Cell. Biol. 2000, 20, 8458-8467. (41) Desterro, J. M. P.; Thomson, J.; Hay, R. T. FEBS Lett. 1997, 417, 297300.

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microelectrosprayed. Calibration solution (Agilent Technologies, Wilmington, DE) was diluted 1:10 into 1:1 water/acetonitrile, 0.1% formic acid and microelectrosprayed from a second identical microelectrospray emitter located in the dual ESI source.42 FT-ICR Mass Spectrometry. Peptide samples were analyzed with a home-built, passively shielded, 9.4-T FT-ICR mass spectrometer43 equipped with an external microelectrospray ionization source.44 The samples were infused at a flow rate of 300 nL/min through an electrospray emitter consisting of a 50-µm-i.d. fusedsilica capillary, which had been mechanically ground to a uniform thin-walled tip.45 A total of 2.0 kV was applied between the microelectrospray emitter and the capillary entrance. The electrosprayed sample and calibrant ions were delivered alternately into the mass spectrometer through a Chait-style atmosphere-tovacuum interface46 and externally accumulated44 for 0.5 s in an rf-only octapole. The ions were transferred through multipole ion guides and trapped in an open47 cylindrical cell (MalmbergPenning trap).48 Ions were frequency-sweep (“chirp”) excited (58-288 kHz, at 150 Hz/µs) and detected in direct mode (512 kword time-domain data). One hundred time-domain data sets were coadded, Hanning apodized, zero-filled once, and subjected to fast Fourier transform (FFT) followed by magnitude calculation. The experimental event sequence was controlled by a modular ICR data acquisition system (MIDAS).49 The FT-ICR mass spectra were internally frequencyto-m/z calibrated50,51 with respect to the calibrant ions. The FTICR mass spectra were analyzed by use of the MIDAS analysis software package.52 Activated-Ion Electron Capture Dissociation and Infrared Multiphoton Dissociation. For tandem mass spectrometry, the peptide ions were externally accumulated44 for 10 or 30 s in an rf-only octapole. A front-end resolving quadrupole53 and/or storedwaveform inverse Fourier transform54,55 ejection served to isolate the peptide ions of interest. (42) Chalmers, M. J.; Quinn, J. P.; Blakney, G. T.; Emmett, M. R.; Mischak, H.; Gaskell, S. J.; Marshall, A. G. J. Proteome Res. 2003, 2, 373-382. (43) Senko, M. W.; Hendrickson, C. L.; Pasa-Tolic, L.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828. (44) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (45) Quinn, J. P.; Emmett, M. R.; Marshall, A. G. In Proc. 46th ASMS Conference on Mass Spectrometry and Allied Topics; Orlando, FL, 1998. (46) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1990, 4, 81-87. (47) Beu, S. C.; Laude, D. A., Jr. Int. J. Mass Spectrom. Ion Processes 1992, 112, 215-230. (48) Malmberg, J. H.; O’Neil, T. M. Phys. Rev. Lett. 1977, 39, 1333-1336. (49) Senko, M. W.; Canterbury, J. D.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1839-1844. (50) Ledford, E. B., Jr.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 27442748. (51) Shi, S. D.-H.; Drader, J. J.; Freitas, M. A.; Hendrickson, C. L.; Marshall, A. G. Int. J. Mass Spectrom. 2000, 195/196, 591-598. (52) Blakney, G. T.; van der Rest, G.; Johnson, J. R.; Freitas, M. A.; Drader, J. J.; Shi, S. D.-H.; Hendrickson, C. L.; Kelleher, N. L.; Marshall, A. G. in Proc. 49th Am. Soc. Mass Spectrom. Conf. Mass Spectrom. Allied Topics; Chicago, IL, 2001. (53) Hendrickson, C. L.; Quinn, J. P.; Emmett, M. R.; Marshall, A. G. in Proc. 49th ASMS Conf. on Mass Spectrometry and Allied Topics; Chicago, IL, 2001. (54) Guan, S.; Marshall, A. G, Int. J. Mass Spectrom. Ion Processes 1996, 157/ 158, 5-37. (55) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897.

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Figure 1. Production of polyubiquitinated GST-Ubc5. Reactions containing ubiquitin, ubiquitin-activating enzyme, and GST-Ubc5 were incubated in the presence of ATP. Samples were removed at the times indicated and the reaction products fractionated by electrophoresis in a 10% polyacrylamide gel containing SDS. The molecular mass of protein markers (kDa) is shown in the right-hand lane.

Figure 2. Direct infusion microelectrospray FT-ICR mass spectrum of an unfractionated tryptic digest of polyubiquitinated GST-Ubc5. The spectrum is internally calibrated with respect to calibrant ions, denoted c. U denotes ubiquitin tryptic peptides. G denotes substrate tryptic peptides.

An indirectly heated dispenser cathode (Heat Wave, Watsonville, CA) mounted on the central axis of the system provided the electrons for ECD.56 A potential of either -2 or -5 V was applied to the cathode during the irradiation event. A grid situated in front of the filament was kept at -200 V for most of the experiment and pulsed to +50 V during the ECD event. An off-axis continuous wave 40 W, 10.6-µm wavelength CO2 laser (Synrad E48-2-115, Bothell, WA) fitted with a beam expander provided the photons for activated ion ECD. The isolated parent ions were irradiated with electrons and photons for 30-50 ms. Between 50 and 100 time-domain data sets were coadded, Hanning apodized, zero-filled once, and subjected to FFT followed by magnitude calculation. The ECD mass spectra were internally frequency-to-m/z calibrated with respect to the precursor ion and the charge-reduced species or backbone fragments. The photons for IRMPD were provided by the laser described above. The isolated parent ions were irradiated with photons for 300-400 ms. Ten time-domain data sets were coadded, Hanning apodized, zero-filled once, and subjected to FFT followed by magnitude calculation. The IRMPD mass spectra were internally frequency-to-m/z calibrated with respect to the precursor ion and backbone fragments. (56) Tsybin, Y. O.; Håkansson, P.; Budnik, B. A.; Haselmann, K. F.; Kjeldsen, F.; Gorshkov, M.; Zubarev, R. A. Rapid Commun. Mass Spectrom. 2001, 15, 1849-1854.

Table 1. Tryptic Peptides Observed in the Direct Infusion MicroESI FT-ICR Mass Spectrum of Polyubiquitinated GST-Ubc5a tryptic peptide

a

residues

charge

measd m/z

calcd m/z

MSPILGYWK MSPILGYW*KIK I*KGLVQPTR GLVQPTR LLLEYLEEK LLLEYLEEKYEEHLYER LLLEYLEE*KYEEHLYER LLLEYLEE*KYEEHLYER YEEHLYERDEGD*KWR YEEHLYER DEGD*KWR KFELGLEFPNLPYYIDGDVK *KFELGLEFPNLPYYIDGDVK FELGLEFPNLPYYIDGDVK LTQSMAIIR YIADKHNM(O)LGGCPK ERAEISMLEGAVLDIR AEISMLEGAVLDIR AEISMLEGAVLDIR IAYS*KDFETLK DFETLK VDFLSK VDFLS*KLPEMLK LPEMLK LPEM(O)LK LPEML*KMFEDR MFEDR RIEAIPQID*KYLK RIEAIPQIDK IEAIPQID*KYLK IEAIPQID*KYLK IEAIPQIDK YL*KSSK SS*KYIAWPLQGWQATFGGGDHPPK YIAWPLQGWQATFGGGDHPPK YIAWPLQGWQATFGGGDHPPK YIAWPLQGWQATFGGGDHPP*KSDLVPR YIAWPLQGWQATFGGGDHPP*KSDLVPR IQ*KWLSDLQR ELSDLQR IAFTTK SQWSPALTVSK E*KYNR EWTQK EWTQ*KYAM

GST-Ubc5 1-9 1-11 10-18 12-18 19-27 19-35 19-35 19-35 28-42 28-35 36-42 45-64 45-64 46-64 65-73 74-87 88-103 90-103 90-103 109-119 114-119 120-125 120-131 126-131 126-131 126-136 132-136 182-194 182-191 183-194 183-194 183-191 192-197 195-218 198-218 198-218 198-224 198-224 242-251 245-251 303-308 327-337 368-372 376-380 376-383

1 2 1 1 1 3 3 2 2 1 1 2 2 2 1 2 2 2 1 2 1 1 2 1 1 2 1 2 1 2 1 1 1 2 3 2 3 2 2 1 1 1 1 1 1

1094.5747 725.3993 1125.6684 770.4515 1149.6423 757.0508 795.0635 1192.0842 1069.9785 1138.5191 1019.4527 1179.1021 1236.1204 1115.0494 1032.5889 781.8866 901.4752 758.9054 1516.8054 714.8711 752.3857 708.3932 767.4081 730.4159 746.4136 761.8739 697.2975 850.9876 1182.6765 772.9374 1544.8671 1026.5870 839.4632 1371.6741 776.0469 1163.5719 1036.5206 1554.2602 672.3644 860.4499 680.3997 1203.6381 823.4206 691.3443 1170.5139

1094.5709 725.4001 1125.6744 770.4525 1149.6407 757.0513 795.0656 1192.0947 1069.9802 1138.5169 1019.4546 1179.1071 1236.1286 1115.0596 1032.5876 781.8766 901.4777 758.9059 1516.8045 714.8724 752.3830 708.3932 767.4212 730.4173 746.4122 761.8736 697.2979 850.9886 1182.6846 772.9381 1544.8688 1026.5840 839.4627 1371.6751 776.0518 1163.5741 1036.5212 1554.2782 672.3678 860.4478 680.3983 1203.6374 823.4062 691.3415 1170.5253

MQIFVK M(O)QIFVK MQIFV*KTLTGK TLTG*KTITLEVEPSDTIENVK TLTG*KTITLEVEPSDTIENVK TITLEVEPSDTIENVK TITLEVEPSDTIENVK IQDKEGIPPDQQR EGIPPDQQR LIFAGK LIFAG*KQLEDGR LIFAG*KQLEDGR QLEDGR TLSDYNIQ*KESTLHLVLR TLSDYNIQ*KESTLHLVLR TLSDYNIQK ESTLHLVLR

Ubiquitin 1-6 1-6 1-11 7-27 7-27 12-27 12-27 30-42 34-42 43-48 43-54 43-54 49-54 55-72 55-72 55-63 64-72

1 1 2 3 2 2 1 2 1 1 2 1 1 3 2 1 1

765.4342 781.4280 690.3899 801.4234 1201.6321 894.4720 1787.9186 762.3943 1039.5198 648.4093 730.8969 1460.7802 717.3557 748.7378 1122.6007 1081.5501 1067.6177

765.4333 781.4282 690.3897 801.4271 1201.6370 894.4675 1787.9278 762.3945 1039.5172 648.4085 730.8967 1460.7861 717.3531 748.7378 1122.6031 1081.553 1067.6213

*K is a Gly-Gly modified lysine residue. M(O) is oxidized methionine.

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Figure 3. Amino acid sequences of GST-Ubc5 and ubiquitin. Underlined regions show sequence coverage observed. Lysines shown in boldface type were observed modified.

RESULTS AND DISCUSSION Polyubiquitinated substrate GST-Ubc5 was generated by incubation of GST-Ubc5 with ubiquitin and the ubiquitin-activating enzyme in the presence of ATP. At the times indicated (see Figure 1), samples were removed and analyzed by SDS-PAGE and Coomassie blue staining. Under these conditions, the GST-Ubc5 undergoes an autoubiquitination reaction forming polymers of ubiquitin linked to GST-Ubc5. After 3 h, the reaction was terminated and the reaction products were digested with trypsin. Figure 2 shows the direct infusion microESI FT-ICR mass spectrum of the tryptic digest of polyubiquitinated GST-Ubc5. The tryptic peptides are detailed in Table 1. The sequences of GSTUbc5 and ubiquitin are shown in Figure 3. The overall sequence coverage obtained for GST-Ubc5 was 55%. However, the sequence coverage included 20 of the 31 total lysine residues in GST-Ubc5. Fifteen modified peptides were observed, revealing that ubiquitination occurred at Lys9, Lys11, Lys27, Lys40, Lys45, Lys113, Lys125, Lys131, Lys191, Lys194, Lys197, Lys218, Lys244, Lys369, and Lys380 (boldface in Figure 3). Most of these residues were observed both modified and unmodified; however, Lys113 and Lys244 were observed as modified only. The overall sequence coverage for ubiquitin was 92%, including six of the seven lysine residues. Four modified peptides were observed, revealing that ubiquitin conjugation occurred at Lys6, Lys11, Lys48, and Lys63 (boldface in Figure 3). Modification of residues Lys27 and Lys33 was not observed. No peptide, either modified or unmodified, containing Lys29 was observed. As these results demonstrate, it may be necessary to employ several digestion procedures, employing various enzymes, to achieve full “lysine coverage” (complete sequence coverage is not required). A further consequence of the experimental procedure, i.e., trypsin digestion, is that it is not clear whether each substrate protein is ubiquitinated at single or multiple sites. Nevertheless, clearly an FT-ICR approach offers advantages for the analysis of ubiquitination in proteins. Analysis time is short because there is no requirement for wet chemical separation. The mass accuracy of the technique allows confident assignment of the modified peptides and, therefore, sites of ubiquitination. To confirm the assignments, tandem mass spectrometry was performed. Figure 4 shows the AI ECD (top) and IRMPD (bottom) FT-ICR mass spectra of doubly protonated TLSDYNIQ*KESTLHLVLR, the modified [55-72] ubiquitin peptide, in which 6986

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Figure 4. AI ECD (top) and IRMPD (bottom) FT-ICR mass spectra of the doubly protonated [55-72] tryptic ubiquitin peptide, TLSDYNIQ*KESTLHLVLR, in which *K is a Gly-Gly modified lysine. Inset: backbone cleavage sites. Both techniques confirm Lys63 as the site of ubiquitin conjugation.

* denotes the Gly-Gly modification of the -amino group of the lysine side chain. The backbone fragments observed following AI ECD and IRMPD of this peptide are shown inset. ECD of peptides and proteins results in cleavage of the N-CR backbone bond to produce c and z• (or c• and z) fragments.57 This fragmentation pathway is unique in tandem mass spectrometry; however, because cleavage occurs at the same backbone linkage (57) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266.

in each amino acid residue, the primary sequence may be deduced. Virtually complete sequence coverage of the modified peptide was observed following AI ECD. Fifteen out of 17 N-CR bonds were cleaved. The Gly-Gly moiety was retained on the backbone fragments, allowing unambiguous identification of the modification site, i.e., Lys63, a well-known site of ubiquitin conjugation. Recent work by Cooper et al.58 revealed that electron capture by -peptides does not result in N-CR cleavage. Additionally, electron capture by synthetic branched peptides, in which branching is achieved through bivalent lysine, does not result in N-C cleavage within the lysine side chain, reinforcing the finding for -peptides. In addition to the production of c, z•, and y ions within the peptide branches, ECD of these branched species results in a characteristic m ion, resulting from cleavage of the modified lysine side chain. The ubiquitination modification emulates the structure of those synthetic branched peptides. In agreement with earlier findings, no cleavage of the N-C bond in the modified lysine side chain was observed in the AI ECD spectrum of [TLSDYNIQ*KESTLHLVLR]2+; however, no m ion, corresponding to the loss of the Gly-Gly modification and the entire lysine side chain, was observed. No fragments arising from cleavage within the modified side chain were seen. IRMPD is a “slow-heating” technique59 in which product ions are formed by the lowest energy pathway(s). IRMPD of peptides and proteins results in cleavage of peptide bonds to produce b and y ions.60,61 Moreover, loss of small neutrals, such as water and ammonia, and internal fragmentation are typically observed. The IRMPD spectrum of the modified peptide shown in Figure 4 (bottom) reveals extensive fragmentation. Fourteen of the 17 peptide bonds were cleaved, slightly less sequence coverage than observed following AI ECD. As for AI ECD, the IRMPD backbone fragments retain the Gly-Gly moiety. Backbone fragments arising from cleavage of both N- and C-terminals to the modified residue were observed; thus, the site of modification may be determined. IRMPD of peptides containing labile modifications typically results in loss of the modification, often at the expense of sequence information. No fragment ions resulting from cleavage within the modification were observed, suggesting that the Gly-Gly modification of the -amino group in lysine is stable. Figure 5 shows the AI ECD (top) and IRMPD (bottom) FTICR mass spectra of the doubly protonated tryptic [7-27] ubiquitin peptide TLTG*KTITLEVEPSDTIENVK, in which * is the Gly-Gly modification of the lysine side chain. The backbone fragments observed following AI ECD and IRMPD of [TLTG*KTITLEVEPSDTIENVK]2+ are shown inset. As in the previous example, virtually complete sequence coverage was observed following AI ECD. Backbone fragments arising from cleavage of 18 of a possible 20 N-CR bonds were observed. c(•) and z(•) fragments arising from cleavage within proline are rarely observed62 due to the cyclic nature of the residue. Backbone cleavage to produce a• and y ions is a minor fragmentation channel in the ECD of peptides. In the present case, y9, i.e., the C-terminal (58) Cooper, H. J.; Hudgins, R. R.; Marshall, A. G. Int. J. Mass Spectrom., in press. (59) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 35, 461-474. (60) Roepstorff, P.; Fohlman, J. Biol. Mass Spectrom. 1984, 11, 601. (61) Biemann, K. Biomed. Environ. Mass 1988, 16, 99-111. (62) Cooper, H. J.; Hudgins, R. R.; Håkansson, K.; Marshall, A. G. Int. J. Mass Spectrom. 2003, 228, 723-728.

Figure 5. AI ECD (top) and IRMPD (bottom) FT-ICR mass spectra of the doubly protonated [7-27] tryptic ubiquitin peptide, TLTG*KTITLEVEPSDTIENVK, in which *K is a Gly-Gly modified lysine. Inset: backbone cleavage sites. Note that the AI ECD cleavage N-terminal to proline is y ion. Lys11 is confirmed as the site of ubiquitination.

backbone fragment arising from cleavage of the peptide bond N-terminal to proline, was observed and the sequence coverage enhanced. No fragment ions arising from cleavage within the modification were observed, and the Gly-Gly moiety was retained on the backbone fragments. The modification site is thus identified unambiguously as Lys11, a less well-known site of ubiquitin conjugation. IRMPD of the same species resulted in backbone fragments arising from cleavage of 18 of the 20 peptide bonds. Again, no fragments resulting from cleavage within the modification were observed, and the modification was retained on the backbone fragments. Fragments b5 and y16, i.e., those arising from cleavage of the peptide bond C-terminal to the modified lysine, were observed; however, cleavage N-terminal to the modified lysine, either b4 or y17, were not. The backbone fragments observed following AI ECD and IRMPD of the doubly protonated substrate [109-119] GST-Ubc5 peptide [IAYS*KDFETLK]2+ (see Figure 6) are shown inset. Extensive sequence coverage was achieved following AI ECD and the Gly-Gly modification was retained on the backbone fragments, allowing unequivocal determination of the site of ubiquitination, i.e., Lys 113. The c7• fragment is particularly abundant in this spectrum. The peak at m/z 842.4413 corresponds to c7 with loss of the Gly-Gly moiety ((m/z)calc 842.4412) via cleavage of the isopeptide bond. Additionally, the peak at m/z 1314.6990 corresponds to y-type cleavage in the modified lysine side chain, i.e., cleavage of the isopeptide bond with loss of the Gly-Gly moiety ((m/z)calc 1314.6945). As noted above, no fragments resulting from Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

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ubiquitinated GST-Ubc5 was generated in vitro and digested with trypsin. The resulting peptide mixture was subjected to directinfusion, i.e., no separation, microESI FT-ICR analysis. The mass accuracy of the FT-ICR technique is such that modified peptides, and therefore sites of ubiquitination, may be identified by mass alone. Fifteen sites of ubiquitination were identified in the substrate protein. Twenty of the total 31 lysine residues were included in the observed peptides. Four sites of ubiquitination were identified within ubiquitin itself, with six of the total seven lysine residues included in the observed peptides. The Gly-Gly moiety is stable and either AI ECD or IRMPD MS/MS is suitable for confirming assignments. Typically, AI ECD resulted in greater sequence coverage than IRMPD. In summary, FT-ICR mass spectrometry offers a number of advantages to the analysis of ubiquitination in proteins. The mass resolution of the technique is such that prior wet chemical separation is not needed. The mass accuracy allows unambiguous assignment of modified peptides as well as sites of ubiquitination. Assignments may be confirmed by IRMPD or AI ECD, with AI ECD, currently unique to FT-ICR, providing the greatest sequence coverage. Figure 6. AI ECD (top) and IRMPD (bottom) FT-ICR mass spectra of the doubly protonated [109-119] tryptic GST-Ubc5 peptide, IAYS*KDFETLK, in which *K is a Gly-Gly modified lysine. Inset: backbone cleavage sites. Both techniques confirm Lys113 as the site of ubiquitination.

N-C cleavage or m ions were observed for the modified residue. IRMPD also resulted in high sequence coverage, in addition to extensive internal fragmentation. IRMPD backbone fragments retained the modification confirming the ubiquitination site as Lys113. No fragment ions arising from cleavage within the modification were observed. CONCLUSION The present results demonstrate the suitability of FT-ICR mass spectrometry for the analysis of ubiquitinated proteins. Poly-

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ACKNOWLEDGMENT The authors thank Drs. Mark R. Emmett and Christopher L. Hendrickson for valuable discussions. The Wellcome Trust (H.J.C.), Cancer Research UK (J.K.H.), and the Biotechnology and Biological Sciences Research Council (R.T.H.) are acknowledged for funding. This work was supported in part by the NSF National High-Field FT-ICR Mass Spectrometry Facility (CHE 9909502), Florida State University, and the National High Magnetic Field Laboratory at Tallahassee, Florida.

Received for review May 26, 2004. Accepted August 30, 2004. AC0401063