Anal. Chem. 2000, 72, 4778-4784
Activated Ion Electron Capture Dissociation for Mass Spectral Sequencing of Larger (42 kDa) Proteins David M. Horn, Ying Ge, and Fred W. McLafferty*
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301
In previous studies, electron capture dissociation (ECD) has been successful only with ionized smaller proteins, cleaving between 33 of the 153 amino acid pairs of a 17 kDa protein. This has been increased to 99 cleavages by colliding the ions with a background gas while subjecting them to electron capture. Presumably this ion activation breaks intramolecular noncovalent bonds of the ion’s secondary and tertiary structure that otherwise prevent separation of the products from the nonergodic ECD cleavage of a backbone covalent bond. In comparison to collisionally activated dissociation, this “activated ion” (AI) ECD provides more extensive, and complementary, sequence information. AI ECD effected cleavage of 116, 60, and 47, respectively, backbone bonds in 29, 30, and 42 kDa proteins to provide extensive contiguous sequence information on both termini; AI conditions are being sought to denature the center portion of these large ions. This accurate “sequence tag” information could potentially identify individual proteins in mixtures at far lower sample levels than methods requiring prior proteolysis.
Sequencing of linear macromolecules such as proteins1 and DNA2 by mass spectrometry (MS) has been of rapidly growing importance since the introduction of matrix-assisted laser desorption (MALDI)3 and electrospray ionization4 (ESI) that can provide mass spectra of such nonvolatile molecules. For proteins, sequence information is derived from the mass values of fragment ions formed by backbone dissociations; a basic limitation is that (1) Biemann, K. In Methods in Enzymology; McCloskey, J. A., Ed.; Academic Press: San Diego, 1990; Vol. 193 p 887. (b) McLafferty, F. W. Acc. Chem. Res. 1994, 27, 379-386. (c) Anderson, J. S.; Svensson, B.; Roepstorff, P. Nature Biotechnol. 1996, 14, 449-457. (d) Qin, J.; Chait, B. T. Anal. Chem. 1997, 69, 4002-4009. (e) Kuster, B.; Mann, M. Curr. Opin. Chem. Biol. 1998, 8, 393-400. (f) McLafferty, F. W.; Kelleher, N. L.; Begley, T. P.; Fridriksson, E. K.; Zubarev, R. A.; Horn, D. M. Curr. Opin. Chem. Biol. 1998, 2, 571-578. (g) Ducret, A.; Von Oostveen, I.; Eng, J. K.; Yates, J. R., III.; Aebersold, R. Protein Sci. 1998, 7, 706-719. (h) McLafferty, F. W.; Fridriksson, E. K.; Horn, D. M.; Lewis, M. A.; Zubarev, R. A. Science 1999, 284, 1289-1290. (i) Jensen, P. K.; Pasa-Tolic, L.; Anderson, G. A.; Horner, J. A.; Lyston, M. S.; Bruce, J. E.; Smith, R. D. Anal. Chem. 1999, 71, 2076-2084. (j) Thomas, J. J.; Bakhtiar, R.; Siuzdak, G. Acc. Chem. Res. 2000, 33, 179-187. (2) Little, D. P.; Aaserud, D. J.; Valaskovic, G. A.; McLafferty, F. W. J. Am. Chem. Soc. 1996, 118, 9352-9359. (3) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (4) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70.
4778 Analytical Chemistry, Vol. 72, No. 20, October 15, 2000
ordering an adjacent pair of amino acids requires at least one mass value resulting from a backbone cleavage between the two residues. In a widely used procedure,1a,c-e,g,j cleavage is effected by extensive sample proteolysis followed by dissociation of molecular ions from the resulting small peptides in the mass spectrometer. However, MS requires orders of magnitude less sample5 than proteolysis, an advantage for the “top down” sequencing approach6 that utilizes only MS, with initial limited cleavage of the protein molecular ion to yield complementary fragment ions representing the whole sequence. Any fragment ion whose mass indicates a structural feature of interest (e.g., posttranslational modification) is dissociated further (MS/MS), with the process repeated (MSn) as necessary.1b,f,g,i,6 A common problem has been that the ion dissociation methods that add energy to induce threshold dissociation of the weakest bonds7-9 do not effect cleavages between all pairs of amino acids for larger than 1-2 kDa peptide ions.1 We report here an improvement for electron capture dissociation (ECD)10,11 to provide extensive terminal sequence information from ECD spectra of proteins such as thiaminase (42 kDa), which is several times larger than those previously measured. ECD is a new MS/MS method that can cause far more and different cleavages,10 in particular the nonergodic dissociation of the amide-N to R-C backbone bond to form c and z‚ products. (5) (a) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1202. (b) Shabanowitz, J.; Settlage, R. E.; Marto, J. A.; Christian, R. E.; White, F. M.; Russo, P. S., Martin, S. E.; Hunt, D. F. In Mass Spectrometry in Biology and Medicine; Burlingame, A. L., Carr, S. A., Baldwin, M. A., Eds.; Humana: Totowa, NJ, 2000. (6) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 21, 806-812. (7) Loo, J. A.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1988, 2, 207-210. Gauthier, J. W.; Trautman, T. R.; Jacobsen, D. B. Anal. Chim. Acta 1991, 246, 211-225. Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 2801-2808. (8) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (9) Price, W. D.; Schnier, P. D.; Williams, E. R. Anal. Chem. 1996, 68, 859866. (10) (a) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (b) Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 2857-2862. (c) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563-573. (11) (a) Kelleher, N. L.; Zubarev, R. A.; Bush, K.; Furie, B. C.; McLafferty, F. W.; Walsh, C. T. Anal. Chem. 1999, 71, 4250-4253. (b) Migorodskaya, E.; Roepstorff, P.; Zubarev, R. Anal. Chem. 1999, 71, 4431-4436. (c) Axelsson, J.; Palmblad, M.; Hakansson, K.; Hakansson, P. Rapid Commun. Mass Spectrom. 1999, 13, 474-477. 10.1021/ac000494i CCC: $19.00
© 2000 American Chemical Society Published on Web 09/21/2000
Figure 1. AI ECD spectrum (50 scans) of ions from ESI of carbonic anhydrase B subjected to in-beam collisions during exposure to lowenergy electrons. The abundance of M29+, M30+, and other (M + nH)n+ ions is ∼1% of the original value. Peaks designated only by mass values could not be assigned to the sequence.
These are also formed from peptides and smaller (95% of the molecular ions, making secondary e- capture probable. Spectra taken at several current values were combined for Figures 2-4, as the higher currents usually produced smaller fragment ions. For example, e- beam currents of 0.10, 0.15, and 0.333 µA gave fragment ions of average size 35, 31, and 23 amino acids, respectively, from thiaminase I (0.05 µA
Figure 4. Cleavages observed in three in-beam AI ECD and one CAD spectrum of thiaminase I, with notation as in Figure 2. Also present in the ECD and CAD spectra were 12 CAD fragmentations between residues 60 and 290.
Figure 5. In-beam AI ECD spectrum (50 scans) of equine cytochrome c.
reduced the molecular ion abundance by only 75% and gave no measurable products). For carbonic anhydrase, 0.2 and 0.333 µA gave c ions of average size 43 and 36, and z‚ ions of 44 and 35, amino acids. In general, more sequence information is obtained by averaging AI ECD scans measured utilizing a range of ecurrent values. Increasing the ion cell pressure (from pulsed gas introduction) for in-beam activation changed primarily the b, y products expected from CAD. An AI ECD spectrum of carbonic anhydrase at 7 × 10-7 Torr cell pressure showed 2 a‚, 7 b, 46 c, 4 y, and 24 z‚ ions, while a spectrum measured under the same conditions except 3 × 10-6 Torr pressure gave 2 a‚, 18 b, 40 c, 15 y, and 25 z‚ ions. Thus, increasing the b and y products by increasing CAD is not important for increasing ECD products, although separate experiments show c, z‚ fragments from ECD of CAD-generated y ions.10c Thus, the cell pressure for AI ECD is not too critical; it is usually adjusted to produce a minor amount of CAD products.
Methods that can be applied to precursor ions mass selected by SWIFT18 (in-beam cannot be used), laser IR,8 and blackbody IR9 irradiation, gave AI ECD for 8.6 and 12.3 kDa proteins, but gave spectra of marginal signal/noise ratios for the 29-42 kDa proteins under the conditions tested. Blackbody IR of carbonic anhydrase ions caused undesirable H2O-loss peaks.20 Preliminary experiments indicate that the IR laser offers promising complementary denaturation, even simultaneously with “in beam”. Carbonic Anhydrase B, 29 kDa. Conventional ECD of the (M + 34H)34+ ions from this protein yielded only the reduced species (M + 34H)31-33+.10c For a mixture of 20+ to 40+ ions entering the ion cell, however, in-beam activation by trapping collisions with N2 gave the AI ECD spectrum of Figure 1, using an electron beam current that reduced the intensity of the parent molecular ions to
