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Probing Protein Tertiary Structure with Amidination Dariusz J. Janecki, Richard L. Beardsley, and James P. Reilly*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A chemical derivatization method, amidination, that has recently been effectively employed in peptide mass spectrometry experiments is used to covalently modify lysines in several standard proteins. Protein and peptide mass spectra identify sites at which the reaction does or does not occur. This is therefore a rapid approach to elucidate solvent-accessible regions of folded proteins. Information about the tertiary and quaternary structure of proteins is critical to understanding enzyme function and mechanism, protein-protein interactions, and protein-ligand binding.1-3 Traditionally, protein structural information is derived using X-ray crystallography or NMR methodologies. To this day, X-ray crystallography is the most powerful technique for structure determination.4,5 However, the throughput of this technique is still rather low due to the difficulty of growing appropriate protein crystals. NMR does not require the growth of crystals, but experiments and their interpretation also take considerable time and the method works best for small proteins.6 Although these powerful biophysical techniques are constantly improving,7,8 intrinsic problems associated with long data collection times, complicated data analysis, and the inability to analyze membrane proteins remain. The Protein Data Bank9 contains ∼28 000 structures (as of 04/ 05/2005), and this number increases by ∼3500 structures per year. Based on homologies and similarities, proteins are grouped into different families having similar folds. The total number of folds and families is still unknown, but some estimates can be given based on complete genome data.10,11 Only 20-30% of the estimated folds have structures available in databases such as SCOP * Corresponding author: (phone) 812-855-1980; (e-mail)
[email protected]. (1) Xu, G.; Chance, M. R. Anal. Chem. 2004, 76, 1213-1221. (2) Glocker, M. O.; Nock, S.; Sprinzl, M.; Przybylski, M. Chem.-Eur. J. 1998, 4, 707-715. (3) Xu, G.; Takamoto, K.; Chance, M. R. Anal. Chem. 2003, 75, 6995-7007. (4) Ban, N.; Nissen, P.; Hansen, J.; Moore, P. B.; Steitz, T. A. Science 2000, 289, 905-920. (5) Liu, H.-L.; Hsu, J.-P. Proteomics 2005, 5, 2056-2068. (6) Christendat, D.; Yee, A.; Dharamsi, A.; Kluger, Y.; Savchenko, A.; Cort, J. R.; Booth, V.; Mackereth, C. D.; Saridakis, V.; Ekiel, I.; Kozlov, G.; Maxwell, K. L.; Wu, N.; McIntosh, L. P.; Gehring, K.; Kennedy, M. A.; Davidson, A. R.; Pai, E. F.; Gerstein, M.; Edwards, A. M.; Arrowsmith, C. H. Nat. Struct. Biol. 2000, 7, 903-909. (7) Norin, M.; Sundstrom, M. Trends. Biotechnol. 2002, 20, 79-84. (8) Abola, E.; Kuhn, P.; Earnest, T.; Stevens, R. C. Nat. Struct. Biol. 2002, 7, 973. (9) http://www.rcsb.org/pdb/. (10) Liu, X.; Fan, K.; Wang, W. Proteins: Struct., Funct., Bioinformatics 2004, 54, 491-499. (11) Wolf, Y. I.; Grishin, N. V.; Koonin, E. V. J. Mol. Biol. 2000, 299, 897-905.
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(Structural Classification Of Proteins12,13) or CATH (Class, Architecture, Topology, Homologous14,15). The SCOP database statistics (release 1.69, July 200516) lists a total of 945 folds and 2845 families. Considering how many proteins are identified and incorporated in databases such as Swiss-Prot or NCBI, it will take many years to uncover the structures and folds of all important disease-related proteins. Another popular method of studying protein structure, solvent accessibility, and protein-ligand interactions is hydrogendeuterium (H/D) exchange.17-22 Slow H/D exchange rates are associated with areas of the biomolecule that are protected while regions that are more exposed to the solvent will show rapid H/D exchange. NMR, IR, or mass spectrometry (MS) can be used to monitor the H/D exchange. Particularly in the past decade, mass spectrometry has displayed several advantages including the ability to detect peptides and proteins with high sensitivity, the ability to study partially exchanged proteins, and the ability to analyze large proteins, either intact or as proteolytic fragments.23 Current H/D exchange technology is limited by the spatial resolution and sequence coverage that can be achieved using NMR or MS. Amide hydrogens that exchange rapidly are bleached out in NMR experiments. Both ESI and MALDI have been used to study H/D exchange in either intact proteins or proteolytic digestion products. Proteolysis is often performed using pepsin at low pH and low temperature to minimize exchange during digestion. The lack of pepsin specificity in enzymatic digestion (12) Hubbard, T. J. P.; Ailey, B.; Brenner, S. E.; Murzin, A. G.; Chothia, C. Nucleic Acids Res. 1999, 27, 254-256. (13) Murzin, A. G.; Brenner, S. E.; Hubbard, T.; Chothia, C. J. Mol. Biol. 1995, 247, 536-540. (14) Pearl, F.; Todd, A.; Sillitoe, I.; Dibley, M.; Redfern, O.; Lewis, T.; Bennett, C.; Marsden, R.; Grant, A.; Lee, D.; Akpor, A.; Maibaum, M.; Harrison, A.; Dallman, T.; Reeves, G.; Diboun, I.; Addou, S.; Lise, S.; Johnston, C.; Sillero, A.; Thornton, J.; Orengo, C. Nucleic Acids Res. 2005, 33, D247-D251. (15) Orengo, C. A.; Pearl, F. M. G.; Thornton, J. M. Methods. Biochem. Anal. 2003, 44, 249-271. (16) http://scop.mrc-lmb.cam.ac.uk/scop/. (17) Englander, J. J.; Del Mar, C.; Li, W.; Englander, S. W.; Kim, J. S.; Stranz, D. D.; Hamuro, Y.; Woods, V. L., Jr. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7057-7062. (18) Hamuro, Y.; Coales Stephen, J.; Southern Mark, R.; Nemeth-Cawley Jennifer, F.; Stranz David, D.; Griffin Patrick, R. J. Biomol. Tech.: JBT 2003, 14, 171-182. (19) Figueroa, I. D.; Russell, D. H. J. Am. Soc. Mass Spectrom. 1999, 10, 719731. (20) Nemirovskiy, O.; Giblin, D. E.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1999, 10, 711-718. (21) Zhu, M. M.; Rempel, D. L.; Zhao, J.; Giblin, D. E.; Gross, M. L. Biochemistry 2003, 42, 15388-15397. (22) Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522-531. (23) Engen, J. R.; Smith, D. L. Anal. Chem. 2001, 73, 256A-265A. 10.1021/ac050891z CCC: $30.25
© 2005 American Chemical Society Published on Web 10/12/2005
creates a large number of peptide possibilities that result in complex data and analysis. Unlike tryptic peptides, many of these will not contain basic sites and will therefore not ionize efficiently. This in turn will often result in lower protein sequence coverage. Another source of complexity in H/D exchange experiments is the appearance of multimodal isotope patterns.19,23 Yet another structural probe with numerous applications is limited proteolysis as described by Hubbard.24 It is based on the hypothesis that proteolysis occurs fastest at “hinges and fringes”25 of a protein. It is expected to take place at flexible, surface loops or, in the case of multidomain proteins, at the mobile linker between the domains. Limited proteolysis has been used to study inhibitory mechanisms of serpins (serine proteinase inhibitors) that are responsible for controlling blood coagulation and inflammation.26 Measuring proteolysis kinetics helps to reveal information about the structure and stability of the cleavable form of a protein.27,28 The application of this technique to the study of native and partially folded protein structures has recently been reviewed.29,30 Protein structure can also be predicted using modern computational techniques. The calculation can be designed so that it becomes an energy minimization problem and a genetic algorithm is used to solve it.31-33 With the development of faster computers, this field of bioinformatics will certainly blossom. Chemical derivatization of accessible sites and further analysis by mass spectrometry has been employed to provide information about the structure of proteins. Przybylski and co-workers34,35 used amino acylation of lysine residues to study surface and binding areas of the translation-elongation factors EF-Tu and EF-Ts 2. Succinylation of lysine residues was likewise employed to map the surface topology of some model proteins.34 Reaction of peptides with hydroxyl radicals generated by γ rays was used to investigate their folding.1,3,36-38 Hydroxyl radicals were also generated by UV irradiation of a 15% hydrogen peroxide solution and used to oxidize specific amino acid chains of two model proteins, lysozyme and β-lactoglobulin A.39,40 Several other labeling approaches have also been employed: amino acylation, tyrosine iodination or nitration.41 Since these methods primarily label (24) Hubbard, S. J. Biochim. Biophys. Acta 1998, 1382, 191-206. (25) Beynon, R. J.; Place, G. A.; Butler, P. E. Biochem. Soc. Trans. 1985, 13, 306-308. (26) Chang, W. S.; Wardell, M. R.; Lomas, D. A.; Carrell, R. W. Biochem. J. 1996, 314 (Pt 2), 647-653. (27) Park, C.; Marqusee, S. Nat. Methods 2005, 2, 207-212. (28) Park, C.; Marqusee, S. J. Mol. Biol. 2004, 343, 1467-1476. (29) Fontana, A.; de Laureto, P. P.; Spolaore, B.; Frare, E.; Picotti, P.; Zambonin, M. Acta Biochim. Pol. 2004, 51, 299-321. (30) Spolaore, B.; Polverino de Laureto, P.; Zambonin, M.; Fontana, A. Biochemistry 2004, 43, 6576-6586. (31) Contreras-Moreira, B.; Fitzjohn, P. W.; Offman, M.; Smith, G. R.; Bates, P. A. Proteins: Struct., Funct., Genet. 2003, 53, 424-429. (32) Juarez, R. G.; Morales, L. B. Rev. Soc. Quim. Mex. 2003, 47, 6-21. (33) Maiocchi, A. Data Handl. Sci. Technol. 2003, 23, 109-139. (34) Glocker, M. O.; Borchers, C.; Fiedler, W.; Suckau, D.; Przybylski, M. Bioconjugate Chem. 1994, 5, 583-590. (35) Suckau, D.; Mak, M.; Przybylski, M. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5630-5634. (36) Xu, G.; Chance, M. R. Anal. Chem. 2005, 77, 4548-4555. (37) Chance, M. R. Biochem. Biophys. Res. Commun. 2001, 287, 614-621. (38) Chance, M. R.; Fiser, A.; Sali, A.; Pieper, U.; Eswar, N.; Xu, G.; Fajardo, J. E.; Radhakannan, T.; Marinkovic, N. Genome Res. 2004, 14, 2145-2154. (39) Sharp, J. S.; Becker, J. M.; Hettich, R. L. Anal. Biochem. 2003, 313, 216225. (40) Sharp, J. S.; Becker, J. M.; Hettich, R. L. Anal. Chem. 2004, 76, 672-683.
Scheme 1. Reaction of S-Methyl Thioacetimidate with Protein Amino Groups
specific residues, they provide less information but the data analysis is less complicated than in H/D exchange experiments.23 It is generally recognized that charged residues arginine and lysine are often preferentially located at protein surfaces.42-48 Exceptions are rather rare.49 Both amino acids are also often involved at active sites, lysine in a binding role and arginine as a participant in catalysis.49 Chemical derivatization of those special amino acids makes them very valuable for probing protein-ligand interactions and mapping protein surfaces. The reaction of imidoesters with proteins and peptides converts primary amines to amidines and has been used in many biochemical studies.50 S-methyl thioacetimidate was reported to yield fewer side reactions at lower pH, and it is a more stable reagent for amidination than O-methyl acetimidate.51 Recently the S-methyl thioacetimidate and thiopropionimidate reagents have been employed to specifically modify lysine residues and N-terminal sites for quantitation of proteins.52,53 In the labeling reaction, the free amino groups of peptide N-termini and lysine side chains are selectively modified (Scheme 1), leading to mass shifts of 41 (with S-methyl thioacetimidate) or 55 Da (with S-methyl thiopropionimidate) per reactive site. Amidination has several advantages over previously investigated lysine modifications. Acylation (e.g., using acetic anhydride) or alkylation by trinitrobenzenesulfonic acid alters both the size and the charge of the amino group. Modifications using succinic anhydride replace a positively charged amino group with a negatively charged carboxyl group.54 These changes may cause partial denaturation and lead to incorrect conclusions about solvent accessibility. Introduction of negative charges may also be problematic for mass spectrometric analysis since molecules will not ionize as well in positive MALDI or ESI experiments. It was reasonable to expect that lysine amidination should be less structurally disruptive, and indeed, the present experiments demonstrate its selectivity for surface exposed sites. (41) Przybylski, M. Adv. Mass Spectrom. 1995, 13, 257-283. (42) Kannan, N.; Schneider, T. D.; Vishveshwara, S. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2000, 56, 1156-1165. (43) Baud, F.; Karlin, S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12494-12499. (44) Karlin, S.; Zhu, Z. Y.; Baud, F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1250012505. (45) Lins, L.; Thomas, A.; Brasseur, R. Protein Sci. 2003, 12, 1406-1417. (46) Karlin, S.; Blaisdell, B. E.; Bucher, P. Protein Eng. 1992, 5, 729-738. (47) Karlin, S.; Brendel, V.; Bucher, P. Mol. Biol. Evol. 1992, 9, 152-167. (48) Karlin, S.; Bucher, P. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 12165-12169. (49) Richardson, J. S.; Richardson, D. C. In Prediction of protein structure and the principles of protein conformation; Fasman, G. D., Ed.; Plenum Press: New York, 1989; p 70. (50) Inman, J. K.; Perham, R. N.; DuBois, G. C.; Appella, E. Methods Enzymol. 1983, 91, 559-569. (51) Thumm, M.; Hoenes, J.; Pfleiderer, G. Biochim. Biophys. Acta 1987, 923, 263-267. (52) Beardsley, R. L.; Reilly, J. P. J. Proteome Res. 2003, 2, 15-21. (53) Beardsley, R. L.; Reilly, J. P. J. Am. Soc. Mass Spectrom. 2004, 15, 158167. (54) Walker, J. M., Ed. The Protein Protocols Handbook; Humana Press: Totowa, NJ, 1996.
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We have investigated the feasibility of using the amidination derivatization of lysine as a probe of a protein’s solvent-accessible surface. Its application could help to quickly answer questions about protein-ligand interactions. For example, the locations and areas of binding sites could be determined. In contrast with H/D exchange, covalent derivatization allows the state of interaction to be frozen. Another attractive aspect of this approach is that the reaction is performed under mild conditions and the native structure of a protein should be retained. As model proteins for this study, we chose well-researched examples of different protein surface structures: ubiquitin (R helix + β sheet), carbonic anhydrase II (all β sheet), and hemoglobin (all R helix).16 Although previous reports of the amidination reaction excluded the possibility of side reactions,51 we also investigated the possibility that other amino acids could be modified. MATERIALS AND METHODS Chemicals. The proteins ubiquitin (U6253), carbonic anhydrase II (bovine, C2522), hemoglobin (human, H7379), bovine trypsin (TPCK treated), and Glu-C endoproteinase were obtained from Sigma (St. Louis, MO). R-Cyano-4-hydroxycinnamic acid (CHCA) and tris(hydroxymethyl)aminomethane (Trizma base, Tris) were also purchased from Sigma. Porcine trypsin modified by methylation (“sequencing grade”) was purchased from Promega (Madison, WI). Anhydrous diethyl ether, thioacetamide, and ammonium bicarbonate were acquired from Fisher (Fair Lawn, NJ). Iodomethane, formic acid, and 2,5-dihydroxybenzoic acid (DHB) were supplied by Aldrich (Milwaukee, WI). Acetonitrile and trifluoroacetic acid (TFA) were obtained from EM Science (Gibbstown, NJ). Derivatization and Tryptic Digestion of Ubiquitin. Ubiquitin was derivatized using S-methyl thiopropionimidate. Reaction mixtures were composed of 100 µM ubiquitin, 0.5 M S-methyl thiopropionimidate, and 1.25 M Trizma base. This mixture was allowed to incubate at room temperature for 1 h before the reaction was terminated by the addition of TFA to a concentration of 3% (v/v). This procedure was also employed for the labeling of denatured ubiquitin. However, in those experiments, a 200 µM aqueous solution of the protein was denatured by incubating for 20 min at 90 °C immediately before mixing ubiquitin with the derivatizing reagents. Prior to tryptic digestion of amidinated ubiquitin the derivatizing reagents were removed using a 3000 molecular weight cutoff filter (Millipore, Billerica, MA). Sequencing grade trypsin (Promega) was employed in both digests. Digestion mixtures were prepared by mixing ubiquitin and trypsin in a 20:1 (ubiquitin/trypsin) molar ratio in an aqueous solution of 25 mM ammonium bicarbonate. Tryptic digestions were incubated for 16 h at 37 °C prior to acidifying with TFA to a concentration of 1% (v/v). Amidination of Bovine Carbonic Anhydrase II (BCAII). A stock solution of 100 µM carbonic anhydrase was prepared in water. A 10-µL aliquot of the stock solution was mixed with appropriate volumes of water and acetonitrile (ACN) to prepare six samples of 200-µL total volumes in which the final concentrations of ACN were 0, 10, 20, 30, 40, and 50% (v/v). Solution of S-methyl thioacetimidate (300 mM) was prepared in 250 mM Tris buffer. A 100-µL aliquot of this amidination solution was added to each sample, and the vials were incubated at room temperature for 2 h. The ACN concentrations in the reaction samples were 0, 7276
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6.7, 13.3, 20, 26.6, and 33.3%, respectively. To stop the reaction and hydrolyze excess reagent, 50 µL of concentrated (96%) formic acid was added to lower the pH to ∼2.5 (checked with pH paper). Samples were then kept frozen at -80 °C until further analysis. All manipulations with amidination reagent were performed in a fume hood. Analysis of Amidinated Ubiquitin. Intact amidinated ubiquitin samples (nondenatured and temperature denatured) were analyzed using MALDI TOF MS. The samples were mixed in 1:9 (v/v) ratio with sinnapinic acid (10 g/L in 50% ACN, 0.1% TFA) matrix solution, and 0.7 µL of the mixture was deposited on a MALDI probe. Digested amidinated ubiquitin was mixed in 1:9 ratio with CHCA (10 g/L in 50%ACN, 0.1%TFA) matrix solution, and 0.7 µL of the mixture was deposited on a probe. All MALDITOF mass spectra of amidinated proteins (intact and digested) were acquired using a Bruker Reflex III reflectron instrument (Bruker Daltonics, Bellerica, MA). Amidination of Hemoglobin. A stock solution of 100 µM human hemoglobin was prepared in water. A 10-µL aliquot of the stock solution was mixed with appropriate volumes of water and ACN to prepare two samples of 200-µL total volumes with 0 and 75% concentrations of ACN (v/v). A 300 mM solution of S-methyl thioacetimidate was prepared in 250 mM Tris buffer. A 100-µL aliquot of this solution was added to the samples and incubated at room temperature for 2 h. The ACN concentrations in the samples were 0 and 50%, respectively. To stop the reaction and hydrolyze excess reagent, 50 µL of concentrated (96%) formic acid was added to lower the pH to ∼2.5 (checked with pH paper). Samples were then kept frozen at -80 °C until further analysis. All manipulations with amidination reagent were performed in a fume hood. Analysis of Amidinated BCAII and Hemoglobin. Amidinated BCAII and hemoglobin samples (∼0.1 µg) were injected onto a Pioneer reversed-phase C4 column, and a gradient was run from 30 to 65% B over 30 min (A, 0.1% formic acid in water; B, 0.1% formic acid in acetonitrile). The effluent was continuously electrosprayed into a QTOF mass spectrometer (Micromass, Manchaster, U.K.). For each sample, the mass spectral scans under the chromatographic peak corresponding to the derivatized protein were combined and deconvoluted. Data acquisition was performed using MassLynx 4.0 software (Micromass) with deconvoluting module MaxEnt3. A volume of 50 µL of each sample was aliquoted and neutralized with enough 250 mM ammonium bicarbonate solution so the pH reached ∼7.0. Sample was dried using a Speedvac and later resuspended in 50 µL of ammonium bicarbonate solution. The volume was split in half, and to each portion either 10 µL (1 µg) of porcine trypsin or Glu-C (V8) enzyme was added and then incubated for 16 h at 37 °C prior to acidifying with TFA to a concentration of 1% (v/v). MALDI-TOF mass spectra of enzymatic digests of amidinated proteins were acquired. The spots using 2,5-DHB were prepared by mixing 1 µL of matrix solution with 0.5 µL of the digests directly on the probe. 2,5-DHB was dissolved in 20% acetonitrile and 0.1% TFA at a concentration of 40 g/L. The MALDI spots using CHCA as matrix were prepared by mixing peptide samples with matrix solution in 1:9 ratio (v/v) and depositing 1 µL of the mixture on the probe. The matrix solution was prepared at 10 g/L concentra-
Figure 2. MALDI-TOF mass spectrum of the tryptic digest of partially amidinated ubiquitin. An inset of the sequence of ubiquitin is included with observed cleavage sites highlighted in boldface type.
Figure 1. MALDI-TOF mass spectra of intact, propionamidinated ubiquitin. (A) Ubiquitin was not denatured prior to reaction. (B) Ubiquitin was thermally denatured before the labeling reaction.
tion in 50% acetonitrile with 0.1% TFA. Each MALDI spot contained ∼200 fmol of digested protein. Signals from 100 laser shots were averaged and internally calibrated using trypsin autolysis peaks.55 RESULTS Mapping Solvent Accessibility of Amine Groups in a Model System: Amidination of Ubiquitin. For the initial test of this labeling method, a small protein, ubiquitin, was studied. This 8565-Da protein contains seven lysines and a free N-terminus that may be derivatized. Although previous work with the amidination of peptides yielded complete reactions at both lysines and N-termini,52 it is possible that the folded tertiary structures of proteins will block the labeling process. The MALDI-TOF mass spectra of Figure 1 demonstrate the labeling efficiency in two different conditions. For the first spectrum (A), ubiquitin was labeled with S-methyl thiopropionimidate under mild conditions (i.e., room temperature and pH 8) that were not expected to induce significant changes to its tertiary structure. Two peaks were observed in this spectrum. The mass of the more intense peak is consistent with the addition of only seven amidine groups whereas the less intense peak was 55 Da heavier, indicating propionamidination of all available sites. For comparison, the native structure of ubiquitin was disrupted by thermal denaturation at 90 °C prior to labeling. As shown in spectrum B, complete derivatization was observed for the unfolded protein. Since the harsh denaturing conditions are expected to disrupt the folded structure of ubiquitin, thereby increasing the solvent accessibility of potential modification sites, these results suggest that the extent of labeling depends on the local environments of the primary amine groups within the folded protein. This effect was further examined by digesting partially amidinated ubiquitin with trypsin to determine which site was not derivatized at room temperature. The tryptic peptides observed in the MALDI-TOF mass spectrum of Figure 2 provide this information. Trypsin specifically cleaves peptide bonds C-terminal (55) Harris, W. A.; Janecki, D. J.; Reilly, J. P. Rapid Commun. Mass Spectrom. 2002, 16, 1714-1722.
Figure 3. Combined and deconvoluted QTOF mass spectra of carbonic anhydrase II amidinated in (A) 0, (B) 6.7, (C) 13.3, (D) 20, (E) 26.6, and (F) 33.3% acetonitrile. Peaks are labeled with the number of lysines amidinated.
to lysine and arginine residues, but amidinated lysines are not susceptible to cleavage. Of the seven lysine residues in this protein only one, Lys27, was involved in an enzymatic cleavage. Two of the observed peptides confirm this. First, the mass of 1832.9 Da matches the peptide composed of residues [28-42] (AKIQDKEGIPPDQQR) in which both lysines were propionamidinated. Furthermore, the mass of 3199.7 Da matches peptide MQIFVKTLTGKTITLEVEPSDTIENVK involving residues 1-27 in which the N-terminus and the first two lysines were modified. Since the tryptic cleavage C-terminal to Lys27 was necessary to produce these two peptides, it is apparent that this residue was not amidinated. Furthermore, the other observed peptides resulted from cleavages adjacent to arginine residues, and no other evidence of cleavage C-terminal to lysine residues was observed. Derivatization of Carbonic Anhydrase II. BCAII was modified using S-methyl thioacetimidate in solutions containing increasing concentrations of acetonitrile (from 0 to 33.3%) as denaturing agent. The modified protein was analyzed by electrospray QTOF MS. Figure 3 shows deconvoluted spectra obtained with solutions having different amounts of ACN. BCAII has 18 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
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Figure 4. Deconvoluted QTOF mass spectra of amidinated human hemoglobin. (A) R chain in water; (B) β chain in water; (C) R chain in 50% ACN; (D) β chain in 50% ACN. Each peak is labeled with the number of amidinated lysines and additional cysteine modifications.
lysines, but its N-terminus is blocked by acetylation and hence cannot undergo reaction with S-methyl thioacetimidate. Seventeen of the 18 lysines were modified when the reaction was performed in water (Figure 3A). Totals of 6.7 and 13.3% ACN yielded similar results (Figure 3B and C). However, with 20% acetonitrile in the amidination mixture, amidination of the last lysine starts to take place (Figure 3D). Essentially complete modification of all 18 lysines was achieved in 26-33% ACN (Figure 3E and F). Some additional peaks can be observed between peaks of amidinated protein. They are 18 Da lighter than the amidinated protein peaks and most likely involve the loss of water. A similar phenomenon is observed with other standard proteins analyzed by QTOF MS. Amidinated bovine carbonic anhydrase was digested with trypsin or Glu-C, and the resulting peptides were analyzed by MALDITOF (data not shown). Observed tryptic peptides resulted from cleavage at arginine with all lysines modified. The N-terminal peptide appeared with acetylation of N-terminal serine and all lysines derivatized. However peptides from the hydrophobic core of the protein (between Asp101 and Asp189) were not observed 7278
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in any MALDI spectra. Overall, we were unable to find the unlabeled lysine using this approach. Amidination of Hemoglobin. The R and β chains of hemoglobin each contain 11 lysines and an N-terminus that is available for amidination. Deconvoluted QTOF mass spectra of hemoglobin derivatized with S-methyl thioacetimidate in 0 and 50% ACN are shown in Figure 4. Each deconvoluted spectrum was obtained by combining mass spectral scans from under chromatographic peaks when the protein was eluting. The R and β chains were resolved chromatographically, but all derivatized versions of each chain eluted together. Several amidinated protein peaks spaced by 41 Da appear in Figure 4A (R chain) and B (β chain). Fully amidinated R chain is expected at 15 619.5Da yet that peak is not observed. Completely amidinated β chain has a calculated mass of 16 360.2Da, but only lower masses can be observed. This means that only a fraction of possible sites are in fact modified when the reaction is performed in water (Figure 4A and B). For both chains, a maximum of 9 of the 12 sites were amidinated (peaks at 15 542.0 Da for R and 16 329.0 Da for β chain). However, most hemoglobin
Figure 5. MALDI-TOF mass spectra of tryptic digestion of hemoglobin amidinated in (A) water and (B) 50% acetonitrile.
molecules were modified at only six (peak 15 418.5Da, R chain) or seven (peak 16 246.5Da, β chain) sites. More complete reaction occurred when hemoglobin was dissolved in 50% acetonitrile (Figure 4C and D). The mass of fully amidinated R chain is 15 619.5Da, and yet one additional peak at 15 666.0Da (46 Da heavier) can also be observed. Similarly, fully amidinated β-hemoglobin should have a mass of 16 360.2, but two peaks above 16 400 Da are observed. As discussed below, these peaks indicate that an additional reaction occurs at cysteine residues. Figure 5 compares MALDI mass spectra of tryptic digests of hemoglobin amidinated in water (Figure 5A) and in 50% acetonitrile (Figure 5B). The two spectra are remarkably different. In the former, peptides resulting from cleavages at arginine (e.g., β[31-40] at 1274.7 Da) and lysine can be observed (e.g., β[1731] at 1529.7 Da). In contrast, the spectrum for hemoglobin
amidinated in organic solvent shows very few peaks. One can be assigned to a peptide with arginine cleavages (1274.73 Da β[3140]). Other intense peaks in this spectrum account for N-terminal peptides of R- and β-hemoglobin with all lysines modified (3318.73 and 3243.85 Da, respectively). There are also two more peaks 41 Da heavier than derivatized N-terminal peptides that result from modification of N-termini. Similar MALDI TOF analysis was performed using Glu-C endoproteinase to digest amidinated samples (data not shown), and the combined data analysis from both digestions led to a sequence map indicating sites that were not amidinated when labeled in aqueous conditions (Figure 6). Additional Modification of Cysteine. As noted above, QTOF mass spectra of amidinated hemoglobin revealed a few unexpected peaks. The leaving group of S-methyl thioacetimidate was found to form a disulfide bond with cysteine residues (Scheme 2). The R and β chains contain one and two cysteines, respectively, leading Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
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Figure 7. NMR structure of ubiquitin with lysine residues highlighted. Figure 6. Sequences of hemoglobin R and β chains. Sites found to be incompletely amidinated are displayed in boldface type and underlined.
Scheme 2. Formation of Methyl Disulfide at Cysteine Residues
to amidinated protein mass shifts of 46 and 92 Da. Each mass spectral peak in Figure 4 is labeled with the number of amine and cysteine sites that are derivatized. Calculated and experimentally observed masses for amidinated hemoglobin are listed in Table 1 of Supporting Information. For direct comparison, a tryptic digest of hemoglobin was treated with amidination reagent and a MALDI TOF mass spectrum was recorded. A cysteine-containing peptide was identified with a 46Da modification. When the same sample was then treated with dithiothreitol, the modified peptide mass disappeared from the spectrum and was replaced by a feature corresponding to the peptide containing reduced cysteine. The spectrum is displayed as Figure 1 in Supporting Information. DISCUSSION Implications of Ubiquitin Results. The observed selectivity of propionamidination in ubiquitin can be explained by considering its native structure. The solution-phase structure (van der Waals radii) of ubiquitin determined by NMR experiments56 (PDB structure 1D3Z) is represented in Figure 7. All lysine residues are shaded dark. With the exception of Lys27, all are exposed on the protein surface and appear to be highly solvent accessible. On the other hand, Lys27 resides in the bottom of a hydrophobic pocket and appears to be relatively shielded. This hindrance most likely explains the incomplete labeling of this site under nondenaturing conditions (Figure 1). Furthermore, the lack of labeling at Lys27 is consistent with previous structural studies involving acetylation of ubiquitin.57 Carbonic Anhydrase II. The zinc enzyme carbonic anhydrase has been extensively investigated and characterized. Bovine carbonic anhydrase is very similar to its counterpart in other (56) Cornilescu, G.; Marquardt, J. L.; Ottiger, M.; Bax, A. J. Am. Chem. Soc. 1998, 120, 6836-6837. (57) Novak, P.; Kruppa, G. H.; Young, M. M.; Schoeniger, J. J. Mass Spectrom. 2004, 39, 322-328.
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mammals (e.g., 87% sequence homology relative to human). According to the crystal structure of BCAII reported in Protein Data Bank (structure 1V9E), it has some helical structure and a dominating β sheet that stretches through the whole molecule. A zinc ion is coordinated to three histidines (His94, His96, His 119). Carbonic anhydrase has been used as a model protein to study the folding and unfolding of polypeptide chains.58-60 One of the advantages in using BCAII is that the protein contains no cysteine residues, thus avoiding constraints associated with disulfide bond formation. Amidination of carbonic anhydrase performed in water resulted in incomplete derivatization. However, in 30% ACN, the protein apparently unfolds and all 18 possible amidination sites are modified. Other research suggests that the equilibrium of unfolding of BCAII occurs through a partly denatured state (molten globule intermediate)58 that preserves much of the secondary structure. Although in our study the hydrophobic core peptides were not detected by MALDI TOF mass spectrometry, it is most probable that in water solution lysine from that hydrophobic core did not react with S-methyl thioacetimidate. H/D exchange data obtained by Jonasson61,62 for human carbonic anhydrase (with tertiary structure similar to BCAII) suggest that residues exchanging through global unfolding are located within the central part of the β sheet that is a component of the large hydrophobic cluster (Thr107-Leu210). Studies of the equilibrium unfolding of BCAII using the ammonium salt of 8-anilinonaphthalene-1-sulfonic acid also suggest unfolding through a transition to a molten globule structure. In this case, unfolding enables hydrophobic surfaces to become accessible to this hydrophobic dye.58,63 Hemoglobin. Crystal structures of the T (tense, low-affinity) and R (relaxed, high affinity) states were first determined some 30 years ago,64 and recently several studies have reported solution structures of hemoglobin obtained with different methods.65,66 Our amidination experiments on hemoglobin in water revealed that many lysines were not derivatized. The quaternary structure (58) Bushmarina, N. A.; Kuznetsova, I. M.; Biktashev, A. G.; Turoverov, K. K.; Uversky, V. N. ChemBioChem 2001, 2, 813-821. (59) Uversky, V. N.; Ptitsyn, O. B. J. Mol. Biol. 1996, 255, 215-228. (60) Ko, B. P. N.; Yazgan, A.; Yeagle, P. L.; Lottich, S. C.; Henkens, R. W. Biochemistry 1977, 16, 1720-1725. (61) Jonasson, P.; Kjellsson, A.; Sethson, I.; Jonsson, B.-H. FEBS Lett. 1999, 445, 361-365. (62) Jonasson, P.; Aronsson, G.; Carlsson, U.; Jonsson, B.-H. Biochemistry 1997, 36, 5142-5148. (63) Rajaraman, K.; Raman, B.; Rao, C. M. J. Biol. Chem. 1996, 271, 2759527600. (64) Perutz, M. F. Nature 1970, 228, 726-734. (65) Ruckebusch, C.; Nedjar-Arroume, N.; Magazzeni, S.; Huvenne, J.-P.; Legrand, P. J. Mol. Struct. 1999, 478, 185-191. (66) He, Y.-F.; Wang, E.-K. Huaxue Xuebao 1997, 55, 801-805.
The two spectra in Figure 5 display remarkably different numbers of peaks. Because trypsin does not cleave at amidinated lysines, fewer peptides arise from the more thoroughly labeled hemoglobin, and some of these are not detected because their high molecular weights yield small MALDI signals. Additional Cysteine Modification. The cysteine modification observed in this work had not been anticipated. As mentioned above our S-methyl reagents lead to formation of a disulfide bond (-S-S-alkyl). Previous reports51 excluded the possibility of reactions involving amino acids other than lysine. While the S-Salkyl formation reaction occurs at free cysteines, already existing disulfide bonds are not affected. This specificity was confirmed by reacting bovine trypsin (that possesses multiple disulfide bonds) with the S-methyl reagent and later analyzing autolytic fragments of trypsin by MALDI MS (data not shown). Figure 8. X-ray crystal structure of hemoglobin. Examples of lysines (dark residues) buried in hydrophobic core (gray residues) of hemoglobin. Three examples of lysines that were not amidinated in aqueous conditions: Lys7, Lys11, and Lys16 from R chain.
of hemoglobin is very tight (structure 1A3N, Protein Data Bank), and many lysines are buried in the hydrophobic core. Organic solvent, such as acetonitrile, facilitates unfolding of the protein, and sites that were buried in the hydrophobic core become accessible to amidination (Figure 8). The N-termini of R and β chains are also not modified by the reagent in water. In nondenaturing conditions, the N-terminus of one chain interacts with the C-terminus of the other in order to create the RβRβ quaternary structure. This interaction tends to bury the N-termini of both chains. Even under denaturing conditions the N-termini of both chains were only partially amidinated (Figure 4C and D), suggesting that even in 50% acetonitrile some residual interactions between N-termini and C-termini still exist. Peptides derived from enzymatic digestions of amidinated proteins were analyzed by MALDI MS using two different matrixes (CHCA or DHB). As discussed previously53 amidinated peptides tend to readily create postsource decay products when CHCA matrix is used (primarily due to neutral loss of NH3). The intensity of neutral loss products was decreased when DHB was used as the matrix. This result is consistent with the well-known observation that 2,5-DHB is a “cooler” matrix.67-69 (67) Karas, M.; Bahr, U.; Strupat, K.; Hillenkamp, F.; Tsarbopoulos, A.; Pramanik, B. N. Anal. Chem. 1995, 67, 675-679.
CONCLUSIONS Reaction of S-methyl thioacetimidate or propionimidate with lysines and the N-terminus of a protein is often incomplete if performed in nondenaturing conditions. Denaturation is found to increase reaction completeness. Although trypsin specifically cleaves peptide bonds C-terminal to lysine and arginine residues, amidinated lysines are not susceptible to cleavage. Tryptic digestion and subsequent analysis by mass spectrometry can therefore reveal which sites are modified and which are not. Although lysines are normally located on the outer surfaces of folded proteins, some may be found in the hydrophobic core and these can be readily identified. A future application for this lysine derivatization method could involve studying protein-protein interactions. The reaction of the reagents with cysteine residues may also have applications in disulfide bond mapping. ACKNOWLEDGMENT This research was supported by the National Science Foundation CHE-0518234, the Indiana Genomics Initiative, and the Indiana 21st Century Fund. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 20, 2005. Accepted September 2, 2005. AC050891Z (68) Gluckmann, M.; Karas, M. J. Mass Spectrom. 1999, 34, 467-477. (69) Brown, R. S.; Carr, B. L.; Lennon, J. J. J. Am. Soc. Mass Spectrom. 1996, 7, 225-232.
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