Anal. Chem. 1999, 71, 4250-4253
Localization of Labile Posttranslational Modifications by Electron Capture Dissociation: The Case of γ-Carboxyglutamic Acid Neil L. Kelleher,§ Roman A. Zubarev,‡ Kristine Bush,† Bruce Furie,† Barbara C. Furie,† Fred W. McLafferty,*,‡ and Christopher T. Walsh§
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, Research East, Beth Deaconess Israel Hospital, Boston, Massachusetts 02115, and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14850-1301
Tandem mass spectrometry (MS/MS) of 28 residue peptides harboring γ-carboxylated glutamic acid residues, a posttranslational modification of several proenzymes of the blood coagulation cascade, using either collisions or infrared photons results in complete ejection of the γ-CO2 moieties (-44 Da) before cleavage of peptide-backbone bonds. However, MS/MS using electron capture dissociation (ECD) in a Fourier transform mass spectrometer cleaves backbone bonds without ejecting CO2, allowing direct localization of this labile modification. Sulfated side chains are also retained in ECD backbone fragmentations of a 21-mer peptide, although CAD causes extensive SO3 loss. ECD thus is a unique complement to conventional methods for MS/MS, causing less undesirable loss of side-chain functionalities as well as more desirable backbone cleavages. INTRODUCTION Despite the development of several methods for tandem mass spectrometry (MS/MS),1 all methods generate similar types of fragment ions by adding energy to the precursor ions to induce threshold dissociation. Thus, while detection of ever more unstable modifications to biomolecules is possible using methods such as electrospray ionization (ESI),2 further microcharacterization of these ionized species with such energetic methods can be difficult as a result of ejection of the modification before backbone bond * Corresponding author. Tel.: 607-255-4699. Fax: 607-255-7880. E-mail:
[email protected]. † Beth Deaconess Israel Hospital. ‡ Cornell University. § Harvard Medical School. (1) Threshold methods for MS/MS: (a) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Sustained Off-Resonance Irradiation. Anal. Chim. Acta 1991, 246, 211-225. (b) Lee, S. A.; Jiao, C. Q.; Huang, Y.; Freiser, B. S. Multiple Excitation Collisional Activation. Rapid Commun. Mass Spectrom. 1993, 7, 819-821. (c) Chorush, R. A.; Little, D. P.; Beu, S. C.; Wood, T. D.; McLafferty, F. W. Surface-Induced Dissociation. Anal. Chem. 1995, 67, 1042-1046. (d) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Infrared Multiphoton Dissociation. Anal. Chem. 1994, 66, 2809-2815. (e) Guan, Z.; Kelleher, N. L.; O’Connor, P. B.; Aaserud, D. J.; Little, D. P.; McLafferty, F. W. 193 nm Photodissociation. Int. J. Mass Spectrom. Ion Processes 1996, 157/158, 357-364. (f) Price, W. D.; Schnier, P. D.; Williams, E. R. Blackbody Infrared Dissociation. Anal. Chem. 1996, 68, 859-866. (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F. Mass Spectrom. Rev. 1990, 9, 37-70.
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cleavage.3,4 An example of such labile species is generated by posttranslational modification of zymogens of serine proteases in the blood coagulation cascade5,6 by the vitamin K-dependent γ-glutamyl carboxylase. The carboxylase uses reduced vitamin K, O2, and CO2 to introduce a CO2 moiety at the γ carbon of glutamic acid residues at the N termini of its protein substrates. These zymogens contain up to 12 γ-carboxyglutamic acid (Gla) moieties within 45 residues.7 Dissection of enzymatic timing and regioselectivity of modification requires cleavage of bonds between these sites in partially carboxylated intermediates; problems with this include the high clustering of such sites and the tendency for decarboxylation in solution. This latter property of Gla residues was utilized in a method using DCl to introduce two γ-deuterium atoms for localization of Gla residues in peptides,8,9 with more direct attempts for this using MS/MS with low- or high-energy collisions resulting in complete decarboxylation before backbone fragmentation.3 Recently, the new MS/MS method of electron capture dissociation (ECD)10,11 for electrosprayed ions cleaves peptide backbones primarily at the CR-N bond rather than at the amide linkage as with collisionally activated dissociation (CAD) or infrared photodissociation (Scheme 1). An early indication that ECD may bypass normal threshold dissociation channels (either by nonergodic fragmentation or a weakened backbone bond in the odd electron ion) came from fragmentation of D2O reacted cytochrome c ions; ECD apparently reduced deuterium atom scrambling, which was extensive with CAD.12 Paralleling this (3) Nakamura, T.; Yu, Z.; Fainzilber, M.; Burlingame, A. L. Protein Sci. 1996, 5, 524-530. (4) Kelleher, N. L.; Nicewonger, R. B.; Begley, T. P.; McLafferty, F. W. J. Biol. Chem. 1997, 272, 32215-32220. (5) Furie, B.; Furie, B. C. In Hematology: Basic Principles and Practice; Hoffman, R. Churchill Livingstone: Edinburgh, 1999; pp 1566-1587. (6) Wu, S. M.; Stanley, T. B.; Mutucumarana, V. P.; Stafford, D. W. Thromb. Haemostasis 1997, 78, 599-604. (7) Gillis, S.; Furie, B. C.; Furie, B.; Patel, H.; Huberty, M. C.; Switzer, M.; Foster, W. B.; Scoble, H. A.; Bond, M. D. Protein Sci. 1997, 6, 185-196. (8) Carr, S. A.; Biemann, K. Biomed. Mass Spectrom. 1980, 7, 172-178. (9) Carr, S. A.; Hauschka, P. V.; Biemann, K. J. Biol. Chem. 1981, 256, 99449950. (10) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (11) 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, 28572862. (12) McLafferty, F. W.; Guan, Z.; Haupts, U.; Wood, T. D.; Kelleher, N. L. J. Am. Chem. Soc. 1998, 120, 4732-4740. 10.1021/ac990684x CCC: $18.00
© 1999 American Chemical Society Published on Web 08/24/1999
Scheme 1
study, an O-glycosylated peptide showed complete retention of the sugars on c ions, some sugar loss from z‚ ions, but complete sugar ejection upon use of threshold dissociation techniques.13 We report here the direct comparison of collisionally activated dissociation (CAD)1a and photodissociation (at 10.6 µm)1d with ECD for peptides containing Gla. These methods are also compared for a sulfated 21-mer peptide. For SO3 and γ-CO2 groups, threshold dissociations result in partial or total loss of these side-chain moieties, whereas fragment ions from ECD retain both. EXPERIMENTAL SECTION All reagents and solvents were obtained from Sigma. The 21mer of sequence KVACLLIKDDKADPNSVTREY was synthesized and purified by HPLC in the Cornell Biotechnology Facility. The Cys residue at position four was sulfated with excess 2-nitro-5thiosulfobenzoic acid (NTSB) using a published procedure,14 and was desalted using a reversed-phase trap. Three peptides of 28 residues having the sequence of residues -18 to +10 of human proprothrombin (proPT28) were synthesized as Glu24-Glu25, Glu24Gla,25 and Gla24-Gla25 proPT28.15 The synthetic products were purified by HPLC, dried, and resuspended in 50:49:1 MeOH/H2O/ AcOH for ESI. The 9.4 T data of Figure 1d,e were acquired from 2 to 4 µL of a 10 pmol/µL peptide solution loaded into a nanospray emitter pulled to a ∼5 µM tip; a platinum wire held at ∼1.5 kV made contact with the solution through the distal end of the emitter. All other data were acquired from ∼200 µL of a 10 pmol/ µL solution infused at 1 µL/min. Multiply charged ions were trapped inside a Fourier transform mass spectrometer (FTMS) at either 616 or 9.4 T,17 with the 6 T instrument adapted for ECD as reported previously.10,11 For MS/MS, ions of interest were isolated by stored waveform inverse fourier transform (SWIFT).18 Ion dissociation by sustained off-resonance irradiation (SORI) was accomplished by pulsed introduction of Ar to 10-6 Torr and application of a frequency ∼1.4 kHz different from that of the precursor ions for 1 s.1a Infrared multiphoton dissociation (IRMPD) used a 40 W CO2 laser and the indicated irradiation times.1d (13) Mirgorodskaya, E.; Roepstorff, P.; Zubarev, R. A., submitted for publication. (14) Thannhauser, T. W.; Konishi, Y.; Scheraga, H. A. Methods Enzymol. 1987, 143, 115-119. (15) Ulrich, M. M.; Furie, B.; Jacobs, M. R.; Vermeer, C.; Furie, B. C. J. Biol. Chem. 1988, 263, 9697-9702. (16) Beu, S. C.; Senko, M. W.; Quinn, J. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1993, 4, 190-192. (17) 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. (18) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (19) All Mr values reported are for the monoisotopic peak.
RESULTS AND DISCUSSION The full ESI/FT mass spectrum of the Glu24-Glu25 proPT28 shows the 6+, 5+, and 4+ charge states generated by ESI (Figure 1a, inset). The relative molecular weight (Mr) value for this peptide was 3361.94 Da,19 a 12 ppm error from that predicted (3361.90 Da; Figure 1a). The two synthetic derivatives of proPT28, with Gla residues in position 25 (Glu24-Gla25 Figure 1b) and in both positions 24 and 25 (Gla24-Gla,25 Figure 1c), show +43.97 (calc. ) 43.99) and +88.02 Da (calc. ) 87.98) increases in their Mr values due to addition of one and two γ-CO2 groups, respectively. Less than 8% of the material has decarboxylated (Figure 1b,c, asterisks), and this could occur in solution and/or in the ESI source.20 To test the stability of the γ-CO2 moiety to MS/MS, the 4+ ions of Gla-Gla-proPT28 (Figure 1c) were isolated by SWIFT and irradiated with infrared photons for 50, 150, and 250 ms (Figures 1d and 1e; 50 ms data not shown). This IRMPD resulted in successive losses of 43.99 Da from the Gla-Gla-proPT28 ions (Figures 1d and 1e). These losses are consistent with ejection of neutral CO2 moieties (-43.99 Da, calc.) most likely from the two Gla residues since such neutral losses of CO2 are not observed in the low-21 or high-energy22,23 fragmentation of multiply protonated peptides. Rather, H2O and NH3 losses such as that of Figure 1e (far left) are far more common. For the 50, 150, and 250 ms spectra, the fragment ions arising from CO2 loss comprise 100, 90, and 75% of the total peak abundances, respectively. This indicates that decarboxylation is the most facile fragmentation pathway and is consistent with randomization of vibrational energy in the precursor ion (i.e., ergodic fragmentation). With 250-ms irradiation, five b- and two y-type fragment ions were observed, including the b18, b23, b24, b25, and b27 and the y25 and y26 fragment ions (summarized in Figure 2d). (The b ions contain the Nterminus, and the y ions contain the C-terminus;24 see Scheme 1.) These fragment ions were all consistent with the predicted sequence of Glu-Glu-proPT28 with no evidence for +44 Da satellites (Figure 1e, arrows in inset), indicating that none had retained the γ-CO2 group of either Gla residue. Further, the b23, b24, and b25 ions directly sequence the two Gla sites, but indicate a Glu24-Glu25 sequence. The γ-CO2 posttranslational modification (PTM) has previously been shown to be ejected before any backbone bond fragmentation of a peptide under both low- and high-energy CAD.3 Thus, neither IRMPD nor CAD can be used to localize Gla in peptides containing Glu, as Gla residues are quantitatively transformed into Glu residues by gas-phase decarboxylation and proton rearrangement (Scheme 2, path a). Some labile PTMs, such as phosphorylation and phosphopantetheinylation, are ejected during MS/MS along with atoms from the protein resulting in a mass “tracer” that allows further localization (e.g., a phosphorylated Ser being converted to dehydroalanine). Here, such a “tracer” strategy could employ 18O, 13C, (20) Detection of γ-carboxyglutamyl residues using Matrix-Assisted Laser Desorption Ionization (MALDI)29 in the positive-ion mode is possible, but spectra acquired for a peptide with two Gla residues in the negative-ion mode resulted in complete decarboxylation upon ionization.3 (21) Senko, M. W.; Beu, S. C.; McLafferty, F. W. Anal. Chem. 1994, 66, 415417. (22) Fabris, D.; Kelly, M.; Murphy, C.; Wu, Z.; Fenselau, C. J. Am. Soc. Mass Spectrom. 1993, 4, 652-661. (23) Kolli, V. S. K.; Orlando, R. J. Am. Soc. Mass Spectrom. 1995, 6, 234-241. (24) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (25) Talbo, G.; Roepstorff, P. Rapid Commun. Mass Spectrom. 1993, 7, 201204.
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Figure 1. ESI/FT mass spectra of synthetic proPT28 peptides with the following residues in positions 24 and 25: (a) Glu24-Glu25 (inset, full mass spectrum); (b) Glu24-Gla;25 and (c) Gla24-Gla.25 All ions have five charges but the scale has been set to mass. The data in (a), (b), and (c) were acquired at 6 T and are single-scan spectra. Infrared photodissociation spectra were acquired after 150 ms (d) and 250 ms (e) of irradiation for the Gla24-Gla25-proPT28 ions in (c); the scale has been set to mass. The data in (d) and (e) were acquired on a 9.4 T instrument and are from single scans. The inset of (e) is an expansion of the 900-990 m/z region of the 250-ms IRMPD spectrum; arrows indicate calculated positions of fragment ions with one or two CO2 groups retained (+44 or +88 Da, respectively).
Scheme 2
or 2H stable isotopes as reported previously,8 but more desirable is the direct localization of the intact Gla residue. Recently, the multiply charged cations from ESI have been shown to capture 90%. Of the 11 fragment ions observed, four have some evidence for CO2 loss (like the c25 ion of Figure 2b), but close inspection of these isotopic distributions (Supporting Information) shows a 45 Da (not 44 Da) loss in Mr value, perhaps due to electron capture at a γ-CO2H group and ejection of CO2H‚ (Scheme 2, bottom right). This 45 Da loss is also observed directly from the intact ions after electron capture. The amount of 45 Da loss here (0-15%) could complicate attempts to assess the regiospecificity of the carboxylase without ambiguity after multiple Glu carboxylations. However, with a sufficient a
Figure 2. Electron capture dissociation spectra of the three following proPT28 derivatives: Glu24-Glu25 (a, 12 scans), Glu24-Gla25 (b, 20 scans), and Gla24-Gla25 (c, 16 scans). These are 6 T data, and the asterisks indicate that a fragment ion has lost 45 Da. The c- and z‚type ions contain the N and C terminus, respectively. The cleavages observed from IRMPD and ECD of proPT28 ions are summarized in (d) and (e), respectively.
small amounts of H2O and H2SO3 loss from the precursor, but negligible such losses from the 12 c and nine z‚ fragment ions (Figure 3b); these provide the complete peptide sequence except for Asp-Pro (but in the CAD spectrum) and for the first four residues that contain the sulfation site. Therefore, SO3 loss is competitive with backbone cleavage during CAD, but an analogous loss of the SO3H moiety during ECD was not observed. Thus, Gla residues can be localized directly by ECD, and to our knowledge, no other MS/MS method is capable of this. Additionally, the ECD methods cleave a greater number of backbone bonds than either collisional or photodissociation, resulting in enhanced sequence information26 and a higher degree of localization of modified residues. Such a gentle and complementary MS/MS method should be of general utility in the localization of labile posttranslational or chemical modifications. Future studies with proteins containing Gla will involve ECD of enzymatically processed substrates. Prior studies have indicated some degree of processivity of the vitamin-K-dependent carboxylase,27 but partially carboxylated protein intermediates are released.28 The kinetic order and regiospecificity of the enzyme for adding 10-12 γ-CO2 groups to its several protein substrates (e.g., profactor IX, profactor X, proprothrombin) can now be addressed efficiently using ECD for localization of Gla residues in the biosynthetic intermediates of serine proteases in the blood coagulation cascade. ACKNOWLEDGMENT The authors gratefully acknowledge Jonathan Tward, Margaret Jacobs, Ted Thannhauser, and Nathan Kruger for experimental assistance and the National Institutes of Health for funding of this research (C.T.W., GM 20011; FWM, GM 16609; B.C.F., HL 42443; K.B., training Grant T32 HL07437; N.L.K., postdoctoral fellowship F32 AI 10087-02). We also thank Chris Hendrickson, Mark Emmett, John Quinn, and Alan Marshall for access to the 9.4 T instrument (National High-Field FT-ICR MS Facility, NHMFL, NSF CHE-94-13008).
Figure 3. Comparison of SORI-CAD (a) and ECD (b) fragmentation spectra (6 T) of 4+ ions of a 21-mer peptide containing a sulfated Cys residue. The peptide structure is shown in (a) (inset), and dots indicate the loss of 80 Da (SO3) from a b-type ion.
signal-to-noise ratio the -45 Da process leaves a mass “tracer” of a Gla residue. Sulfation (R-S-SO3H) is also a posttranslational modification whose site localization by threshold dissociations may be compromised by facile SO3 loss.25 For a 21-residue peptide, the Mr value of 2457.26 Da (data not shown) was consistent with that calculated for the monosulfated product (2457.19 Da). Its CAD spectrum of (M + 4H)4+ has dominant fragment ions for the losses of H2O and SO3 and eight b-type and two y-type ions (Figure 3a). The b ions containing the sulfated Cys have mostly retained the SO3 group, but satellite peaks correspond to the loss of 79.96 Da (solid circles). The b10 (small) and b13 fragment ions that still contain the SO3 group only restrict its location to half of the peptide. The ECD spectrum of the same (M + 4H)4+ ions has
NOTE ADDED IN PROOF In-source decay of MALDI ions has been proposed to be an example of ECD.11 ISD also appears to retain labile side-chain modifications such as those from phosphorylated serine and threonine residues. SUPPORTING INFORMATION AVAILABLE Isotopic distributions of the c26 and c24 fragment ions of Figure 3. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 21, 1999. Accepted July 6, 1999. AC990684X (26) McLafferty, F. W.; Fridriksson, E. K.; Horn, D. M.; Lewis, M. A.; Zubarev, R. A. Science (Washington, D.C.) 1999, 284, 1289-1290. (27) Morris, D. P.; Stevens, R. D.; Wright, D. J.; Stafford, D. W. J. Biol. Chem. 1995, 270, 30491-30498. (28) Stanley, T. B.; Wu, S.; Houben, R. J. T. J.; Mutucumarana, V. P.; Stafford, D. W. Biochemistry 1998, 37, 13262-13268. (29) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A.
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