Anal. Chem. 2003, 75, 3263-3266
Electron Capture Dissociation and 13C,15N Depletion for Deuterium Localization in Intact Proteins after Solution-Phase Exchange Jay P. Charlebois, Steven M. Patrie, and Neil L. Kelleher*
Department of Chemistry, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
For localization of deuterium atoms after solution-phase exchange with D2O, intact proteins are often digested prior to analysis by mass spectrometry (MS) and tandem MS (MS/MS). Amelioration of limitations associated with this approach (e.g., 3 kDa offers improved diversity of backbone sites cleaved when compared to the exhaustive use of “threshold” techniques, such as infrared multiphoton dissociation (IRMPD) and collisionally activated dissociation (CAD) of the same ions.16,17 Although the exact mechanism(s) of ECD has yet to be delineated fully, a recent empirical result is that labile posttranslational modifications (PTMs) normally ejected during threshold MS/MS experiments (e.g., γ-carboxylation of Glu) are preserved during the ECD process.18 The gentle nature of MS/MS by ECD produces vibrationally cold fragment ions from greater backbone sites and led us to extend ECD of intact protein ions to D atom localization after H/D exchange in solution. Recently, direct fragmentation of intact proteins after H/D exchange in a Fourier transform mass spectrometer (FTMS) has been demonstrated using CAD in the nozzle skimmer region11 or in a hexapole used for external accumulation.18 However, the D content of individual amide sites along the peptide backbone could not be determined.19 In addition, given that PTMs that are labile during threshold MS/MS of peptides tend not to be at the protein level,19,20 it is reasonable that H/D scrambling could be a (12) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 35, 461-474. (13) Akashi, S.; Naito, Y.; Takio, K. Anal. Chem. 1999, 71, 4974-4980. (14) Demmers, J. A. A.; Rijkers, D. T. S.; Haverkamp, J.; Killian, J. A.; Heck, A. J. R. J. Am. Chem. Soc. 2002, 124, 11191-11198. (15) Zubarev, R.; Kelleher, N.; McLafferty, F. J. Am. Chem. Soc. 1998, 120, 3265-3266. (16) Zubarev, R.; Horn, D.; Fridriksson, E.; Kelleher, N.; Kruger, N.; Lewis, M.; Carpenter, B.; McLafferty, F. Anal. Chem. 2000, 72, 563-573. (17) Zubarev, R. A.; Haselmann, K. F.; Budnik, B.; Kjeldsen, R.; Jensen, F. Eur. J. Mass Spectrom. 2002, 8, 337-349. (18) Kelleher, N.; Zubarev, R.; Bush, K.; Furie, B.; Furie, B.; McLafferty, F.; Walsh, C. Anal. Chem. 1999, 71, 4250-4253. (19) Akashi, S.; Takio, K. Protein Sci. 2000, 9, 2497-2505. (20) Kelleher, N. L.; Nicewonger, R. B.; Begley, T. P.; McLafferty, F. W. J. Biol. Chem. 1997, 272, 32215-32220.
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function of precursor ion size as well as local sequence effects.13,14 Avoiding such complications by fragmentation out of far lower vibrational states, ECD shows promise as a method to eliminate all amide-H/D scrambling during MS/MS and to probe the microenvironments of individual amide sites directly from species >5 kDa. Here, we demonstrate this H/D-ECD approach for the first time and quantify the improvements in c and z• ion signals obtained in the direct fragmentation approach (no proteolysis) through depletion of 13C and 15N heavy isotopes.21,22 EXPERIMENTAL METHODS Yeast Recombinant Ubiquitin. A plasmid carrying the yeast ubiquitin gene and coding for a His tag on the C-terminal end was provided by Professor George Makhatadze (Penn State University).23 The plasmid was used to transform the BL21(DE3)pLysS strain of competent cells (Promega). The transformed cells are inducible by isopropyl-β-D-thiogalactopyranoside (IPTG). Both the normal and depleted yeast ubiquitin grown for these experiments were grown in 100-mL batches in either Luria broth or Mono-express cell growth medium (12C 99.95%, 14N, 99.97%, Cambridge Isotope Laboratories). Cells were grown at 37 °C. Protein overexpression was induced with IPTG at OD 0.4 at λ 600 nm and allowed to continue overnight. Protein isolation and purification was performed using Ni-NTA resin (Qiagen) and following the manufacturer’s protocol. The protein was purified by HPLC (0.5% formic acid in both organic and aqueous mobile phases) and dried prior to analysis by mass spectrometry. Upon detecting mass discrepancies in the protein product by ESI/FTMS and MS/MS, the gene sequence was determined at the W. M. Keck Center for Comparative and Functional Genomics (University of Illinois). After DNA resequencing, the revised protein sequence (Figure 3) yielded 8 sequence discrepancies and a corrected theoretical relative molecular weight (Mr) value of 9336.87-0 Da (with Start Met). Mr values are reported as monoisotopic unless indicated otherwise with an italicized number of heavy isotope peaks. Deuteration of Yeast Recombinant Ubiquitin. Purified protein was resuspended in 400 µL of D2O (pH meter reading ∼5.8) to a final concentration of 1.2 µM and was maintained at room temperature for the duration of the exchange period. Fiftymicroliter aliquots were removed and frozen in liquid nitrogen at 30-min intervals. Frozen specimens were thawed, desalted and electrosprayed into the mass spectrometer as described below. Any portion of the desalted protein that was not actively being analyzed was refrozen immediately. Widths of isotopic distributions after H/D exchange were determined by counting the number of isotopic peaks that were above 5% abundance relative to the most abundant peak. FT-ICR-MS and ECD. The deuterated protein was then rapidly analyzed via FTMS after SWIFT isolation. The electrospray solution was sprayed from a 50-µm-i.d. emitter (New Objective) at 1 µL/min from an acetonitrile/water/acetic acid solution (78: 18:2). The voltage on the electrospray emitter was held at 2200 (21) Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S.; Guan, S.; Logan, T. M. J. Am. Chem. Soc. 1997, 119, 433-434. (22) Akashi, S.; Takio, K.; Matsui, H.; Tate, S.-i.; Kainosho, M. Anal. Chem. 1998, 70 (15), 3333-3336. (23) Loladze, V.; Ermolenko, D.; Makhatadze, G. Protein Sci. 2001, 10, 13431352.
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Figure 1. The 9+ charge state from the broadband ESI-FT-ICRMS spectra of (a) nondepleted recombinant yeast ubiquitin (UB6HLB; 10 scans) and (b) 13C,15N doubly depleted recombinant yeast ubiquitin (UB6H-DEP; 5 scans). Insets, expansions of isotopic distributions for the +56-Da forms of the protein observed.
Figure 2. ECD spectra of and expanded views of (a) nondepleted recombinant yeast ubiquitin (100 scans) and (b) 13C,15N depleted recombinant yeast ubiquitin (100 scans). The insets show an expanded portion of the spectra and the S/N levels of selected peaks.
V, with a heated metal capillary aiding ion desolvation at the instrument inlet. Ions were accumulated for 2-4 s in an accumulation octopole prior to injection into the FTMS cell. For ECD, 200250 pulses of low-energy (
