Identification of Protein Ubiquitylation by Electrospray Ionization

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Anal. Chem. 2006, 78, 3681-3687

Identification of Protein Ubiquitylation by Electrospray Ionization Tandem Mass Spectrometric Analysis of Sulfonated Tryptic Peptides Dongxia Wang,†,‡,§ Dario Kalume,‡,| Cecile Pickart,⊥ Akhilesh Pandey,| and Robert J. Cotter*,|

Department of Pharmacology and Molecular Sciences, Department of Biological Chemistry, Johns Hopkins University School of Medicine, and Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205

Covalent conjugation of ubiquitin to cellular proteins is an important posttranslational modification. Protein ubiqutylation signals proteolysis, as well as nonproteolytic functions.1-3 Ubiquitin-mediated processes regulate a number of biological cellular

events including cell cycle, DNA repair and transcription, protein quality control, and immune response. A trienzyme system is involved in ubiquitin conjugation that includes the ubiquitinactivating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-ligase (E3). Ubiquitin conjugates to specific target proteins through the covalent linkage between the carboxyl group of its C-terminal glycine and the side chain of a lysine residue within the substrate protein via an isopeptide bond. Advances in mass spectrometry have made it possible to directly identify ubiquitin-modified proteins and to determine the sites of ubiquitylation. Diglycine branched peptides are produced upon the digestion of ubiquitin-conjugated proteins by trypsin, where the cleavage of ubiquitin’s C-terminal RGG moiety leave a Gly-Gly branch on those peptides that contain ubiquitylation sites. Based on the tandem mass spectrometric analysis of tryptic peptides where the Gly-Gly branch is treated as a small modifier, Marotti and co-workers reported the direct identification of an ubiquitylation site in a G protein subunit by tandem mass spectrometry.4 Peng and co-workers described a proteomic approach to globally detecting protein ubiquitylation from Saccharomyces cerevisiae cells and identified 110 ubiquitylation sites in 72 ubiquitin-protein conjugates.5 Cooper and co-workers developed an accurate mass-based approach using Fourier transform ion cyclotron resonance mass spectrometry for the analysis of a model ubiquitinated protein.6 Borchers’ group investigated diagnostic fragment ions for protein ubiquitylation with model ubiquitinated tryptic and Glu-C peptides.7,8 We have recently reported a novel strategy for identifying ubiquitylation sites using chemical derivatization and matrixassisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry.9 N-Terminal sulfonation of tryptic peptides

* To whom correspondence should be addressed. E-mail: [email protected]. † Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine. ‡ These authors contributed equally to this work. § Current address: Biotechnology Core Facility, National Center for Infectious Disease, Centers for Disease Control and Prevention, Atlanta, GA 30333. | Department of Biological Chemistry, Johns Hopkins University School of Medicine. ⊥ Johns Hopkins University Bloomberg School of Public Health. (1) Hershko, A.; Ciechanover, A. Annu. Rev. Biochem. 1998, 67, 425-479. (2) Pickart, C. M. Annu. Rev. Biochem. 2002, 70, 503-533 (3) Weissman, A. M. Nat. Rev. Mol. Cell Biol. 2001, 2, 169-178.

(4) Marotti, L. A., Jr.; Newitt, R.; Wang, Y.; Aebersold, R.; Dohlman, H. G. Biochemistry 2002, 41, 5067-5074. (5) Peng, J.; Schwartz, D.; Elias, J. E.; Thoreen, C. C.; Cheng, D.; Marsischky, G.; Roelofs, J.; Finley, D.; Gygi, S. P. Nat. Biotechnol. 2003, 21, 921-926. (6) Cooper, H. J.; Heath, J. K.; Jaffray, E.; Hay, R. T.; Lam, T. T.; Marshall, A. G. Anal. Chem. 2004, 76, 6982-6988. (7) Warren, M. R.; Parker, C. E.; Mocanu, V.; Klapper, D.; Borchers, C. Rapid Commun Mass Spectrom. 2005, 19, 429-437. (8) Parker, C. E.; Mocanu, V.; Warren, M. R.; Greer, S. F.; Borchers, C. H. Methods Mol Biol. 2005, 301 153-173. (9) Wang, D.; Cotter, R. J. Anal. Chem. 2005, 77, 1458-1466.

We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carried out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitinconjugated proteins using LC/MS/MS.

10.1021/ac051904b CCC: $33.50 Published on Web 05/05/2006

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adds two sulfonic acid tags to the N-terminus of the peptide and its Gly-Gly branch, respectively. The MS/MS analysis of such peptide derivatives results in a specific fragmentation distribution that includes a signature portion that distinguishes these branched peptides from other modified and unmodified peptides and a sequence portion that directly reveals the amino acid sequence of the modified peptide and the location of the ubiquitylation site by a series of y-type product ions. In this way, ubiquitinated peptides can be readily determined. The method has been demonstrated with model peptides and proteins and has been used successfully in identifying ubiquitylation sites in ubiquitinconjugated C-terminal Hsc70-interacting protein.10 In the present work, we describe the application of the N-terminal sulfonation method to the electrospray ionization (ESI) tandem mass spectrometric analysis of ubiquitin-conjugated proteins using model peptides and proteins. Electrospray mass spectra are generally dominated by multiply charged ions, and others have shown that multiply charged sulfonated peptides produced by ESI undergo a different fragmentation mechanism and result in a fragment distribution pattern different from singly charged derivatives.11 Thus, differences in the fragmentation behavior of doubly tagged species might be expected as well. In addition, the low-energy collision-induced dissociation (CID) used in the ESI QTOF mass spectrometer can be distinctively different from the laser-induced (or postsource) dissociation used in the MALDI TOF/TOF mass spectrometer.12 EXPERIMENTAL SECTION Materials. All chemicals used in this study were of analytical grade. 4-Sulfophenyl isothiocyanate (SPITC) and ammonium bicarbonate were purchased from Sigma (St. Louis, MO). Bovine pancreas modified trypsin was supplied by Roche Diagnostics Corp. (Indianapolis, IN). R-Cyano-4-hydroxycinnamic acid was from Aldrich (Milwaukee, WI). All synthetic peptides were synthesized and purified by the Synthesis & Sequencing Facility at the Johns Hopkins University School of Medicine (Baltimore, MD). Tetraubiquitin (Ub4) was purchased from Affinity Research Products, Ltd. (Exeter, UK). Ubiquitin-conjugated proteins, Ub3dihydrofolate reductase (DHFR) and Ub5-DHFR, were prepared as previously described.13 Sample Preparation. The synthetic peptides (∼1-10 pmol) were derivatized by mixing 1 µL of peptide with 4 µL of SPITC (10 mg/mL in 20 mM NaHCO3, pH ∼9.0) and incubating for 30 min at 55 °C. The reaction was terminated by adding 1 µL of 1% TFA. For protein samples, 4 µg of Ub4, Ub3-DHFR, or Ub5DHFR protein was digested with trypsin (20:1 w/w) in 10 µL of the reaction solution containing 25 mM ammonium bicarbonate at 37 °C for 18 h. A 5-µL sample of the reaction was then mixed with 5 µL of SPITC solution and derivatized following the same sulfonation procedure. The solutions were diluted 20-fold by deionized water prior to mass spectrometric analysis. (10) Wang, D.; Xu, W.; McGrath, S. C.; Patterson, C.; Neckers, N.; Cotter, R. J. J. Proteome. Res. 2005, 4, 1554-1560. (11) Keough, T.; Youngquist, R. S.; Lacey, M. P. Anal. Chem. 2003, 75, 157165A. (12) Rosario, M.; Domingues, M.; S.-Marques, M. G. O.; Carla, A. M.; Vale, C. A. M.; Neves, M. G.; Cavaleiro, J. A. S.; Ferrer-Correia, A. J.; Nemirovskiy, O. V.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1999, 10, 217-223. (13) Thrower, J. S.; Hoffman, L.; Rechsteiner, M.; Pickart, C. M. EMBO J. 2000, 19, 94-102.

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Sample micropurification and preparation for analysis of the derivatized synthetic peptides by NanoESI-MS/MS was performed as described.14 Samples were purified using a reversed-phase homemade nanocolumn (GeLoad tip-Eppendorf). The peptides were acidified by adding formic acid to 5% (v/v) final concentration and loaded on to the nanocolumn packed with Poros Oligo R3 reversed-phase chromatography medium (10-µL bed volume) (Applied Biosystems, Foster City, CA) and previously equilibrated with the same solution. A washing step using 5% formic acid solution was carried out and approximately four bed volumes were passed through the column. Afterward, 150 µL of 90% acetonitrile (v/v) solution was loaded on to the column, and the peptides eluted in a 0.5-µL microcentrifuge tube. The solution was dried down to remove the organic solvent, redissolved in 20 µL of acetonitrile-1% formic acid (1:1 v/v). Mass Spectrometry. The purified peptide solution was loaded on to a silver-coated glass capillary emitter. The NanoESI-MS/ MS mass spectra of the synthetic peptides were acquired with different collision energies (20, 30, and 40 eV). The tryptic peptide mixtures derived from the protein digestion were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/ MS). The mixtures (10 µL) were loaded on-line onto a fused-silica capillary column packed with 5-µm Vydac C18 resin. The mobile phase used for gradient elution consist of (A) 100% H2O with 0.4% acetic acid and 0.005% heptafluorobutyric acid and (B) 90% acetonitrile with 10% H2O, 0.4% acetic acid, and 0.005% heptafluorobutyric acid. The peptides were separated using a linear gradient elution from 10 to 45% B in 40 min followed by 45-90% B in 3 min. A potential of 2.5 kV was applied to the emitter in the ion source. The spectra were acquired on a Micromass Q-TOF APIUS (Manchester, U.K.) equipped with an ion source sample introduction system designed at Proxeon Biosystems (Odense, Denmark). The acquisition and the deconvolution of data were performed on a MassLynx Windows NT PC data system (version 4.0). All spectra were obtained in the positive-ion mode. RESULTS AND DISCUSSION To study the application of N-terminal sulfonation method for ubiquitinated peptides by electrospray ionization, we first examine the fragmentation of singly charged precursors using the Q-TOF mass spectrometer with an electrospray ion source. A synthetic peptide containing the Gly-Gly branch, LK(GG-)FAGAQLEDGR, was derivatized and mass analyzed. The peaks corresponding to the singly and doubly charged states of the sulfonated peptide with the addition of two sulfonic acid tags, to the N-termini of the peptide and the branch, were observed in the MS spectrum. As shown in Figure 1, the MS/MS analysis of the singly charged precursor of the peptide derivative produces a spectrum whose fragmentation pattern is similar to those observed previously for diglycine branched peptide derivatives by MALDI MS/MS.9,10 The product ions can be divided into two distinctive groups, the signature and sequence portions. The high-mass fragments (from m/z 1248.62 to 1633.67) correspond to the loss of the sulfonic acid tags and the first N-terminal residues at both the peptide and the branch. This group of product ions forms a signature pattern unique to the Gly-Gly branched peptide. The sequence (14) Gobom, J.; Nordhoff, E.; Mirgorodskaya, E.; Ekman, R.; Roepstorff, P. J. Mass Spectrom. 1999, 34, 105-116

Figure 1. ESI MS/MS spectrum of the SPITC sulfonation derivative of the peptide, LIK(GG-)AGAQLEDGR. The spectrum was obtained from singly charged precursor. A y sequence ion series up to y11 is readily observed. Sequence ions that have lost tag-L and tag-G correspond to the signature pattern of the mass spectrum.

portion of the spectrum (from m/z 175.11 to 1305.65) consists of a series of y-type product ions that directly reveal the amino acid sequence of the peptide. The mass difference between y10 and y11 corresponds to that of a diglycine-modified lysine residue and determines the location of the ubiquitylation site within the peptide. Compared to MALDI mass spectra of similar derivatives, several slight differences were observed. First, the relative intensities of the peaks corresponding to the elimination of one tag (m/z 1633.67) and two tags (m/z 1418.73) were much lower relative to the other signature ions, that is: fragmentation of the tag along with its immediate amino acid residue was nearly as facile under collision-induced dissociation conditions. Second, while signature ion peaks showed much higher intensities than those corresponding to sequencing ions in MALDI MS/MS spectra, the relative intensities of the fragment ions in the sequencing portion are comparable to those of the signature ions. With these minor differences between the spectra of MALDI and ESI, the formation of a unique fragmentation pattern for diglycine branched peptides, analyzed as their N-terminal-sulfonated derivatives, provides a strategy that can be used equally well in ESI and MALDI MS/MS analysis. Electrospray ionization produces multiply charged ions as well where the doubly charged ions are the most prominent within the size range of tryptic peptides. In this study, a doubly charged precursor was also formed for the same synthetic branched, sulfonated peptide. Using a collision energy of 30 eV, a typical energy for the instrument for the tandem mass spectrometric analysis of tryptic peptides, the distribution of the product ions observed (Figure 2A) was distinctly different from the one generated from the singly charged precursor. Both singly and doubly charged product ions were observed, and despite Nterminal derivatization, other types of product ions, such as b ions, were formed along with a complete series of y ions. Indeed, the fragmentation of doubly charged peptides involves a more complicated mechanism than that proposed for singly charged sulfonation derivatives9 and does produce a considerably more

complicated mass spectrum. The suppression of b-ion formation resulting from an N-terminal negative charge would not be the case for a fragment ion carrying two positive charges. Therefore, the potential advantage of the N-terminal sulfonation approach to the formation of y ions exclusively is not realized for doubly charged precursors. Three of the high-mass signature peaks (m/z 1520.62, 1305.67, and 1248.63) are observed, but their lower intensities reduce their usefulness in identifying Gly-Gly branched peptides. To enhance the formation of characteristic product ions for doubly tagged peptide derivatives, we performed CID experiments on the doubly charged species under increased or decreased collision energy. With higher collision energy at 40 eV, no obvious changes occurred in the spectrum shown in Figure 2B. Three peaks at m/z 232.14, 845.41, and 916.44 increased in their relative intensities but correspond to the ions y2, y8, and y9, respectively, and are not signatures for the sulfonic acid tags and diglycine branch. At lower collision energy (20 eV), however, significant changes were observed in the fragment ion distribution (Figure 2C). The relative intensities of the three most intense fragments, one singly charged ion at m/z 329.06 and two doubly charged ions at m/z 624.81 and 760.80, were more than 3-fold higher than these of all other peaks. The singly charged peak at m/z 1520.62 increased as well. The significance of these changes in the fragmentation pattern under various experimental conditions can be appreciated in the deconvoluted spectra (Figure 2D-F), where all product ions are plotted as their singly charged species. The three most intense peaks (P2, P3, P4) and the highest mass peak (P1) in Figure 2F represent fragments directly related to the sulfonation tags and the diglycine branch. As before, the peaks P2 (m/z 1520.62) and P3 (m/z 1248.61) represent the fragments resulting from the loss of tag-Leu and the elimination of both tag-Leu and tag-Gly groups from the precursor ion, respectively. The peak P1 (m/z 1576.65) corresponds to the release of the tag plus glycine residues from the branch while the peak P4 at m/z 329.06 represents the b1 fragment (tag-Leu). Although the fragments P2Analytical Chemistry, Vol. 78, No. 11, June 1, 2006

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Figure 2. MS/MS spectra of doubly charged sulfonation derivative of the diglycine branched peptide, LIK(GG-)AGAQLEDGR. The spectra were generated at the collision energy of (A) 30, (B) 40, and (C) 20 eV. Their deconvoluted spectra were shown in (D), (E), and (F), respectively.

P4 and the fragments P3 and P4 also appeared in the spectra obtained at collision energies of 30 (Figure 2A,D) and 40 eV (Figure 2B,E), the distinctive intensities of these fragments only appeared in the low-energy CID spectra (Figure 2C,F). The formation of characteristic fragments from sulfonation derivatized branched peptides can be explained by the mechanism 3684

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called “Edman”-type preferential cleavage proposed by Gaskell and co-workers15,16 and reported by other laboratories including (15) Summerfield, S. G.; Bolgar, M. S.; Gaskell, S. J. J. Mass Spectrom. 1997, 32, 225-231. (16) van der Rest, G.; He, F.; Emmett, M. R.; Marshall, A. G.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 2001, 12, 288-295.

Figure 3. Effect of the diglycine branch positions on the fragmentation of sulfonated peptide derivatives, (A) LIFK(GG-)GAQLEDGR, (B) LIFAGAQLK(GG-)DGR, and (C) LIFAGAQLEDK(GG-)R. The deconvoluted spectra were obtained from doubly protonated precursor at the collision energy of 20 eV.

ours.9,17 The nucleophilic attack of the thiocarbonyl group in the sulfonation tag, SPITC in the present work, on the carbonyl group of the N-terminal peptide bond was believed to promote the cleavage of the peptide bond between the first and the second N-terminal amino acid residues. The strong nucleophilicity of the thiocarbonyl group makes the first peptide bond weaker than others and causes the preferred cleavage under fragmentation conditions. For the sulfonated branched peptides, the preferential fragmentation under low collision energy occurred at two peptide bonds; one is the first N-terminal peptide bond and another is the bond between two glycine residues in the branch, resulting in four related fragments. It is reasonable to speculate that only two characteristic fragments might be generated under the same condition from a sulfonated linear peptide. The observation of the product ion of tag-Leu but not tag-Gly reveals a weak bond between SPITC tag and the first glycine residue within the diglycine branch, presumably due to glycine’s nonhindered side chain. In general, the mass of the P4 peak can be found within the range from 272 to 401 where the former value is the mass sum of the SPITC tag and the smallest residue, glycine, and the latter one is the sum of the tag and the largest amino acid residue, tryptophan. The mass range of the other signature peaks will also be similarly constrained within a region close to the molecular ion. These features will be helpful in reducing false positive identifications for ubiquitinated peptides. Another intrinsic feature of the characteristic ions is that the mass difference between the peaks P2 and P3 is a constant, 272 (from singly charged ions) or 135.5 (from doubly charged product (17) Lee, Y. H.; Kim, M.-S.; Choie, W.-S.; Min, H.-K.; Lee, S.-W. Proteomics 2004, 4, 1684-1694.

ions), corresponding to the mass of tag-Gly. It should be noted that this is true as well for the MS/MS spectra (see Figure 1) of a singly charged peptide derivative, where the difference in mass between the peak at m/z 1248.62 and the peak at m/z 1520.59 gives rise to the constant of 272. In addition to using this constant to confirm positive identifications by manual inspection of MS/ MS spectra, it could also be utilized to facilitate automatic selection of the signature pattern-containing spectra of ubiquitinated peptides with an appropriately designed computer routine. The development of such an automated approach is ongoing in our laboratories. Although the fragmentation of doubly charged precursors under high energy was not able to generate a characteristic pattern for derivatized branched peptides, the dissociation into a series of well-distributed product ions enhanced our ability to obtain unambiguous amino acid sequencing and to map the modification site. Despite the occurrence of a nearly complete series of y ions in the spectra shown in Figure 2C and F, low-energy CID might not always generate sufficient product ions for the complete and unambiguous recovery of peptide sequence. In practice, a lowenergy MS/MS could be carried out on derivatized peptide digests as a type of survey scan for modified peptides, followed by a second MS/MS at higher collision energy on these precursors to obtain the sequence information. On the other hand, the product ion at m/z 141.10 observed in the spectra (Figure 2A, B, D, and E) under high CID conditions is in fact a marker ion for Gly-Gly modified peptides. The mass of the ion corresponds to the loss of ammonia from the immonium ion (158.18 Da) of one glycine-modified lysine residue. Increasing the collision energy up to 70 eV did not disrupt this marker ion, suggesting a stronger Analytical Chemistry, Vol. 78, No. 11, June 1, 2006

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Figure 4. (A) Total ion chromatogram of sulfonated peptide mixtures digested from Ub5-DHFR by trypsin. (B) MS/MS spectra of the peptide at m/z 945.81 (2+) at 20 eV.

bond between the glycine residue and the side chain of the modified lysine residue (data not shown). Thus, this marker ion for glycine-modified lysine residues added an additional parameter for identification and confirmation of ubiquitin-modified peptides. To examine the effect of the branch position on the formation of signature fragments, three synthetic branched peptides with the Gly-Gly modification at different positions, LIFK(GG-)GAQLEDGR, LIFAGAQLK(GG-)DGR, and LIFAGAQLEDK(GG-)R, were prepared and investigated. All MS/MS spectra (Figure 3) were acquired at a CID energy of 20 eV from doubly charged precursors of N-terminal sulfonated peptides. As expected, all of the spectra displayed the characteristic pattern 3686

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containing the four characteristic fragments, indicating that the signature pattern was not affected by the location of diglycine branch within target analytes. The feasibility of the present strategy was further demonstrated by the analysis of three ubiquitin-conjugated proteins, Ub4, Ub3DHFR, and Ub5-DHFR. Ub4 is a tetraubiquitin where four ubiquitins are connected to each other through their K48 residues. Ub3-DHFR and Ub5-DHFR are two model substrates for 26S proteasomes, where di- and tetraubiquitin chains are in vitro conjugated to a ubiquitin-DHFR fusion protein.11 As in Ub4, the N-terminal fused ubiquitin is modified at its K48 residue. The protein conjugates were first digested by trypsin and followed by

N-terminal sulfonation with SPITC. The peptide mixtures were then subjected to LC/MS/MS processing with 20 eV of collision energy. Not surprisingly, the peptide bearing a Gly-Gly branch at lysine 48 residue, LIFAGK(GG-)QLEDGR, digested from the conjugates, was readily identified by the signature pattern from all of the three samples. A typical total ion chromatogram and an MS/MS spectrum of the target peptide obtained from Ub5-DHFR, respectively, are shown here in Figure 4A and B. These results indicate that the sulfonation method combined with tandem mass analysis by electrospray ionization mass spectrometry can be effectively utilized in the characterization of protein ubiquitylation, an important posttranslational modification. CONCLUSION We described here a novel approach for characterizing posttranslation protein ubiquitylation using N-terminal sulfonation and electrospray ionization mass spectrometry. While the tandem mass analysis of a singly charged precursor results in a fragment ion distribution similar to that observed by MALDI mass spectrometry, the MS/MS of the doubly charged precursor of a sulfonated target produces a relatively different signature fragmentation pattern under low-energy CID conditions. The fragmentation pattern is itself sensitive to the collision energy,, and in this case, the lower collision energy of 20 eV produced an increase in the signature ions relative to the sequencing ions. This provides a

convenient means for rapidly screening peptide fragments for ubiquitylation, but it should also be noted that, within the broad dynamic range of the mass spectrometer, a complete set of y-ions with good signal/noise could also be observed and used for locating the ubiquitylation site. The method was developed with several synthetic diglycine branched peptides and was demonstrated by three ubiquitin-conjugated proteins. Overall, our research indicates that the sulfonation strategy for identifying ubiquitylated tryptic peptides can be used in two of the most common mass spectrometry configurations. Characterization of proteins modified by ubiquitin from more complex sample systems using this new method is ongoing in our laboratories. ACKNOWLEDGMENT This work was supported by a contract BAAHL-02-04 from the National Heart Lung and Blood Institute (Jennifer Van Eyk, PI) and a grant U54RR020839 from the National Institutes of Health (Jef Boeke, PI). Mass spectral analyses were carried out at the Middle Atlantic Mass Spectrometry Laboratory.

Received for review October 25, 2005. Accepted March 8, 2006. AC051904B

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