Use of Isotopic Signatures for Mass Spectral Detection of Protein

Oct 12, 2005 - Jisook Kim , Albert R. Vaughn , Chris Cho , Titus V. Albu , Ethan A. Carver. Bioorganic ... Diganta Sarma , Robert P. Hanzlik. Journal ...
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Chem. Res. Toxicol. 2005, 18, 1748-1754

Use of Isotopic Signatures for Mass Spectral Detection of Protein Adduction by Chemically Reactive Metabolites of Bromobenzene: Studies with Model Proteins Weimin Yue,† Yakov M. Koen, Todd D. Williams, and Robert P. Hanzlik* Department of Medicinal Chemistry, University of Kansas, Malott Hall, Room 4048, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045 Received July 20, 2005

The cytotoxicity of many small organic compounds often apparently derives from their metabolic activation and covalent binding to cellular proteins. It is therefore of considerable interest to be able to determine, for a given protoxin, which metabolites modify which proteins at which sites. Our laboratory has identified more than 45 target proteins for bromobenzene metabolites in liver by peptide mass mapping after two-dimensional electrophoresis. Through all of this work, we have never observed a bromine-containing peptide. We therefore generated model adducted proteins by carbodiimide coupling of NR-acetyl-Nτ-(p-bromophenyl)-L-histidine (1) and NR-acetyl-N-(p-bromophenyl)-L-lysine (2) to bovine pancreatic ribonuclease A. For the adducts, RNase-(1)n and RNase-(2)n, mass spectrometry indicated that n ) 0-2 and 0-6, respectively. RNase-(2)n was submitted to in-gel and in-solution digestion with trypsin, and the digests were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDITOF) and liquid chromatgraphy electrospray ionization MS (LC/ESI-MS) and tandem MS (MS/MS). Sequence coverages observed ranged from 67% with only three modified lysines observed using in-gel digestion and MALDI-TOF analysis, to 100% coverage with all 10 lysines observed in both modified and unmodified form using in-solution digestion and LC/ESI-MS. In the mass spectra of all modified peptides up to 2000 Da, the bromine isotope pattern was obvious by visual inspection; for peptides up to 3600 Da, the isotopic signature could be recognized by visual comparison to simulated spectra. The presence of Br-containing adducts was confirmed by MS/MS analysis of selected peptides. The selection of peaks for MS/MS analysis was significantly facilitated by visual recognition of the bromine isotope pattern, even at signal-to-noise ratios of 10 (or lower in favorable cases). These results indicate that stable isotope labeling may have considerable potential for detecting and locating protein adducts of reactive metabolites.

Introduction Many simple organic molecules containing phenyl substituents or benzene rings become cytotoxic upon biotransformation to reactive electrophilic metabolites (1-4). Prime examples include acetaminophen, diclofenac, and bromobenzene (BB).1 Their cytotoxicity is strongly correlated with covalent binding of reactive metabolites to various cellular proteins. Key questions concerning cellular injury by reactive metabolites are the identities of the target proteins, the residues within them that become covalently modified, and the functional consequences of these covalent modifications. As a start toward elucidating the detailed biochemical mechanism(s) of BB hepatotoxicity, we identified the structures of adducts of BB metabolites to rat liver proteins by hydrolyzing unfractionated proteins and elucidating the structures * To whom correspondence should be addressed. Tel: 785-864-3750. E-mail: [email protected]. † Current address: School of Chemistry and Pharmacy, East China University of Science and Technology, Meilong Road 130, P.O. Box 363, Shanghai 200237, China. 1 Abbreviations: BB, bromobenzene; BBO, bromobenzene-3,4-oxide; CID, collision-induced dissociation; DTT, dithiothreitol; ESI, electrospray ionization; LC, liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MMCO, molecular mass cutoff; TOF, timeof-flight; SDS, sodium dodecyl sulfate.

of modified amino acids released; most arose from the alkylation of cysteine residues by quinone metabolites (5-7), but we also found that bromobenzene-3,4-oxide (BBO), thought to be the primary “toxic” metabolite of BB (8), alkylates histidine and lysine as well as cysteine residues of rat liver proteins, albeit to a lesser extent (9). In other work, we identified several rat liver proteins targeted by BB metabolites. One was a nonspecific esterase (10) previously found to be a target for metabolites of halothane and molinate (11, 12). Surprisingly, other prominent BB target proteins included epoxide hydrolase2 and glutathione transferases (13), which are supposed to detoxify BBO. In continuing this work, we have now identified (13, 14) a total of 46 target proteins for reactive BB metabolites from various rat liver subcellular fractions.3 Most of these identifications have been based on peptide mass mapping of tryptic digests of proteins labeled in vivo with [14C]BB, separated by twodimensional (2D) electrophoresis, and located by autoradiography. Remarkably, in all of this work involving hundreds of peptide identifications, we have not observed a single peptide that was adducted by a bromine2 3

Rombach, E. M., and Hanzlik, R. P. Unpublished results. Koen, Y. M., and Hanzlik, R. P. Unpublished results.

10.1021/tx050199z CCC: $30.25 © 2005 American Chemical Society Published on Web 10/12/2005

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experiments. In addition, the data show that even at peptide masses approaching 2000, the isotope pattern or “signature” from a single bromine atom is sufficient to make recognition of bromine-containing peptides relatively easy by visual inspection alone. We believe this augurs well for the use of synthetically introduced stable isotope signatures for detecting, within a digest mixture, peptides bearing reactive metabolite adducts. Figure 1. Sequence of bovine RNase A with lysine residues shown in boldface.

containing metabolite of BB. This is despite the fact that bromophenyl-cysteine, -lysine, and -histidine can be isolated from total acid hydrolysates of these proteins (5, 7, 9) and detected in intact rat liver proteins by western blotting after one-dimensional (1D) polyacrylamide gel electrophoresis (PAGE) (15, 16). There could be several possible reasons for our failure to observe bromine-containing peptides in digests of proteins from BB-treated rat liver: (i) a majority of the covalent binding may involve metabolite(s) that have lost the bromine atom; (ii) a generally low level of total adduction may occur, such that adducted peptides are a very small fraction of the total digest mixture; (iii) within any given protein molecule, adduction may occur at multiple sites in parallel, such that any single adducted peptide is a minor component of a complex mixture; (iv) the observed peptides in a digest often represent incomplete coverage across the entire protein sequence; or (v) a combination of these factors may operate to make the observation of adducted peptides especially difficult. The situations characterized above may be contrasted to the example of analyzing the adduction of an enzyme by an active site-directed affinity labeling reagent or a suicide substrate. In such cases, the stoichiometry of labeling is usually high, approaching one adducted residue per molecule of protein, and adduction usually involves a single active site residue, yielding a single modified peptide in a digest. The analytical challenges noted above are not unique to proteins adducted by BB metabolites, and they apply equally well to the analysis of proteins labeled in vivo by any type of chemically reactive metabolite, of which there are many (4, 17-19). To investigate the reasons that we have not detected bromine-containing peptides from in vivo samples, we decided to generate some bromine-containing model proteins, analyze them by proteolytic digestion and mass spectrometry, and use our findings to assess and perhaps improve the analytical procedures that we apply to real proteins from in vivo experiments under toxicologically relevant conditions. We have previously found bovine RNase (Figure 1) to be useful as a model for other studies of protein adduction by reactive metabolites (20) and for haptenization to create target antigens for enzyme-linked immunosorbent assay analyses (15, 16). Thus, we constructed model adducted proteins by N-ethyl-N′-(3-diethylaminopropyl)carbodiimide hydrochloride (EDC) coupling of 1 and 2 to lysine side chains on RNase and analyzed the conjugates [RNase-(1)n and RNAse-(2)n, respectively] and their tryptic digests by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) and electrospray ionization (ESI) mass spectrometry. In this manuscript, we report results demonstrating that we can indeed observe bromine-containing peptides, even in small samples (,50 pmol), in both MS and MS/MS

Experimental Procedures Materials. NR-Acetyl-Nτ-(p-bromophenyl)-L-histidine (1) and NR-acetyl-N-(p-bromophenyl)-L-lysine (2) were prepared as described previously (21, 22). Bovine pancreatic ribonuclease A (RNase) was from Sigma and was used without further purification. Sequencing grade trypsin was obtained from Roche (Nutley, NJ). Coupling of 1 and 2 to RNase. Compound 1 (28.2 mg, 80 µmol) was dissolved in DMSO (1.5 mL), and 0.1 M sodium phosphate buffer (1 mL, pH 5) was added with stirring. EDC (60 mg, 320 µmol) was added as a single portion, and the mixture was stirred at room temperature for 10 min and then added to a solution of 50 mg of ribonuclease A predissolved in 0.2 M sodium phosphate buffer (8 mL, pH 9). The resultant milky solution was stirred at room temperature for 18 h, more EDC (30 mg, 160 µmol) was added, and stirring was continued for another 24 h. The resulting solution was dialyzed [molecular mass cutoff (MMCO) 6000-8000 Da] against 2.0 L of deionized water at 4 °C (six changes, 12 h/change). After dialysis, the solution was centrifuged to remove particulates (10 min at 8000 rpm), and the clear supernatant was lyophilized to give RNase(1)n as a white solid (30 mg). Similar coupling of 2 (27.4 mg, 80 µmol) to RNase afforded 31 mg of lyophilized RNase-(2)n. Electrophoretic Analysis of Modified RNase. Electrophoretic separation was performed using a Mini-PROTEAN II cell equipped with a 1000/500 Power Supply (Bio-Rad Laboratories). RNase samples were separated by tricine-sodium dodecyl sulfate (SDS)-PAGE according to a reported method (23). A separating gel with an acrylamide concentration of 16.5% (3% Bis) and a 4% stacking gel were used. The gels were run at 30 V for 30 min and then at 90 V (increasing to 150 V to keep the current constant at 15 mA) for another 180 min until the dye front disappeared. The proteins were visualized using 0.025% Coomassie brilliant blue R250 in a mixture of methanol/ acetic acid/water (50:10:40, v/v/v) followed by destaining in 10% acetic acid. Whole Protein Analysis by MALDI-TOF-MS and ESIMS. Lyophilized samples of RNase-(1)n and RNase-(2)n were redissolved in water. For MALDI-TOF analysis, the solution was mixed with a solution of sinapinic acid (matrix) and applied to a sample plate from Voyager-DE STR mass spectrometer (Perseptive Biosystems, Framingham, MA). The latter was operated in positive linear mode (accelerating voltage, 25 kV; extraction delay, 750 ns). Data acquisition was performed over the m/z range 2000-60000. Mass spectra were calibrated by the default method and verified by unmodified RNase as internal calibration. ESI spectra were acquired on a Q-TOF2 (Micromass Ltd., Manchester, United Kingdom) hybrid mass spectrometer operated in MS mode and acquiring data with the TOF analyzer. The instrument was operated for maximum sensitivity with all lenses optimized while infusing a sample of lysozyme. The cone voltage was 60 eV. Spectra were acquired at 11364 Hz pusher frequency covering m/z 800 to 3000 and accumulating data for 3 s per cycle. Time to mass calibration was made with CsI cluster ions acquired under the same conditions. Samples were desalted on a short column (3 cm × 1 mm i.d.) of polymeric HPLC resin (Hamilton PRP1, Reno, NV) by loading the sample in 1% HOAc and eluting protein with 95% MeOH and 0.08% formic acid at 25 µL/min directly into the source. In-Solution Digestion with Trypsin. RNase-(1)n and RNase-(2)n (380 µg, 27.7 nmol) were dissolved in a digestion

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solution containing 8 M urea and 0.4 M NH4HCO3 (0.6 mL). After brief sonication to dissolve the samples, duplicate 40 µL samples of each solution were transferred into 250 µL Eppendorf tubes containing 10 µL of dithiothreitol (DTT) solution (54 mM in H2O) and the mixture was incubated at 50 °C for 15 min. After it was cooled to room temperature, 4-vinylpyridine solution was added (10 µL, 100 mM in 100 mM aqueous NH4HCO3) and the reaction mixture was held at room temperature for 20 min in the dark. Next, water (30 µL) and a solution of sequencing grade trypsin (7.5 µg of trypsin dissolved in 30 µL of H2O) were added to achieve a final enzyme:protein ratio of 1:85 (w/w), and the final solution was incubated at 37 °C for 18 h. The reaction was then frozen for later analysis by HPLC/MS. In-Gel Digestion with Trypsin. RNase-(1)n and RNase-(2)n samples were individually separated using tricine-SDS-PAGE (23), and the relevant bands (∼14 kDa) were excised from the gel. The gel pieces were submitted to two cycles of washing with 200 mM NH4HCO3 in 50% aqueous acetonitrile (150 µL) for 30 min at 30 °C followed by dehydration with pure acetonitrile (150 µL) for 30 min. The dehydrated gel pieces were then heated for 60 min at 50 °C with 50 µL of DTT solution (10 mM in 100 mM NH4HCO3), which was then replaced with 4-vinylpyridine solution (50 µL, 2% v/v in 100 mM NH4HCO3 solution), and the mixture was kept at room temperature for 45 min in the dark. Gel pieces were then submitted to two cycles of washing (100 mM NH4HCO3 solution, 150 µL) and dehydration (150 µL of CH3CN) and finally air-dried. For digestion, the pieces were rehydrated briefly in 20 µL of a solution containing trypsin (12 ng/µL) and CaCl2 (5 mM) in 50 mM NH4HCO3 solution, after which 40 µL of 5 mM CaCl2 in 50 mM NH4HCO3 solution was added and the mixture was incubated overnight at 37 °C. After it was vortexed and centrifuged, the supernatant was transferred to a vial and 10% aqueous trifluoroacetic acid (TFA) was added to make the final concentration 1% (v/v). As a control, protein-free (blank) regions of gel were processed similarly. MALDI-TOF-MS Analysis of Tryptic Digests. Digests were concentrated in a C18 Zip-tip and eluted with 2 µL of a saturated solution of R-cyano-4-hydroxy-trans-cinnamic acid in 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid and applied to a sample plate. Samples were analyzed on a Voyager-DE STR MALDI-TOF-MS operated in positive reflector mode with an accelerating voltage of 20 kV, a mirror voltage ratio of 1.12, and an extraction delay of 180 ns. Data acquisition was performed over the m/z range 700-3000. Mass spectra were externally calibrated using a standard mixture of known peptides covering the entire mass range, where possible the calibration was verified using mass peaks arising from trypsin autolysis. HPLC/ESI-MS Analysis of Tryptic Digests. Tryptic peptides were separated on a RP-HPLC column (0.32 mm i.d. × 5 cm Zorbax SBC18, 300 Å pore size, 3.5 µm particles packed by Micro-Tech Scientific, Vista, CA) at a flow rate of 10 µL/min with a linear gradient from 20 to 95% (v/v) methanol in 0.08% (v/v) aqueous formic acid using an Ultra Plus II MicroLC system (Micro-Tech). Peptides were directly eluted into the source of a Q-TOF2 mass spectrometer with Ar admitted to the collision cell, and data were acquired in TOF mode limiting spectra to 200-2500 u in 5 s acquisition cycles. Collision-induced dissociation (CID) spectra were acquired with automatic function switching between survey MS and MS/MS modes on ions with intensity of >3 counts per second and found in a look-up table. The latter was populated with masses found by manual searching of the liquid chromatography (LC)/MS data for the appearance of “bromine-like” isotope clusters. The Q1 precursor selection window was set to transmit 5 u to capture all bromine isotopomers. Argon was the collision gas, and the collision energy was 25 eV.

Results Analysis of Whole Proteins. Commercial RNase was examined by conventional SDS-PAGE and found to

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Figure 2. Whole molecule mass spectra of RNase haptenized with 1 or 2. (A) MALDI mass spectrum of RNase-(1)n; (B) ESI mass spectrum of RNase-(1)n; (C) MALDI mass spectrum of RNase-(2)n; and (D) ESI mass spectrum of RNase-(2)n. In each spectrum, the peaks are numbered to indicate the number of adduct moieties attached.

contain only relatively small amounts of contaminating proteins. After subjecting the RNase to coupling with EDC-activated 1 or 2 and reisolating the protein, reexamination by SDS-PAGE showed no discernible changes as compared to untreated RNase (data not shown). Examination of treated RNase samples by mass spectrometry, however, showed clear evidence for modification to differing extents. After treatment of RNase with EDC-activated 1, MALDI-TOF-MS indicated the product [i.e., RNase-(1)n] to be a mixture of RNase molecules containing 0-2 molecules of covalently bound 1 (Figure 2A). This was confirmed by separate analysis using ESIMS (Figure 2B), which shows three sharp peaks separated by 334 ( 1 amu, the expected mass increment for each addition of 1 (with loss of water for amide bond formation). The decreasing peak abundance with increasing modification is consistent with statistical expectations for a limited degree of reaction of a monovalent reagent with a protein having 11 potential sites of reaction (i.e., 10 relatively similar lysine residues plus an unblocked R-amino group at the N terminus). Previous studies of the acylation of RNase in solution have shown that except

Adduct Recognition by Isotopic Signatures

at very low pH or with extremely large excesses of acylating reagent, acylation occurs exclusively at lysine side chains (24). In agreement with this expectation, we have observed no modified peptides that did not contain at least one missed cleavage (see below). The coupling of 2 to RNase under identical conditions led to a much more extensively modified protein [RNase(2)n] as shown by both MALDI-TOF-MS and ESI-MS (Figure 2C,D, respectively). Again, the spacing of the mass peaks (325 ( 1 amu) corresponds to stepwise addition of 2 with elimination of water for amide bond formation. The much greater degree of RNase modification by activated 2 vs activated 1 under identical coupling conditions probably reflects the fact that as an acylating agent, activated 2 is less hindered than activated 1. The nearly statistical distribution of products is also noteworthy. However, it must be remembered that there are potentially 11 isobaric isomers of monohaptenized RNase and an even greater number of isomers for various multiply adducted protein molecules. Analysis of Protein Digests. The RNase-(1)n and RNase-(2)n conjugates were submitted to electrophoresis under standard 1D tricine-SDS-PAGE conditions. Appropriate regions of the gel were excised and subjected to in-gel digestion as described in the Experimental Procedures, and the digests were analyzed by MALDITOF-MS and LC/ESI-MS. For comparison, small samples of haptenized RNase (i.e., amounts comparable to the 3-5 µg contained in a typical gel band) were submitted in parallel to in-solution digestion and the digests were analyzed similarly. Our objectives were to determine whether we could (i) obtain sequence coverage as good as that obtained with unmodified RNase, (ii) observe adducted peptides containing missed cleavages caused by lysine modification, and (iii) observe the 79Br/81Br isotopic signature above the background isotope pattern due to 13C and other natural isotopes as the molecular mass increased up to 2000 and beyond. If stable isotope signatures could be relied upon for recognition of adductcontaining peptide peaks in tryptic digests, it would be very beneficial to proteomic and mechanistic studies of reactive metabolites, not only those with a naturally occurring signature such as BB but also for those with artificially introduced isotopic signatures. Under the conditions that we used for tryptic digestion, the formation of large peptides containing missed cleavages is ordinarily a very minor process; typically, less than 10% of the peptides in a digest contain missed cleavages. However, a large or pKa-modifying substitution on a lysine side chain, such as acylation by 1 or 2 or even acetic anhydride (24), blocks tryptic cleavage at that lysine, resulting in the appearance in the digest of new peptides that are both longer and adduct bearing. A proline residue following an unmodified lysine, such as Lys41-Pro42 in unmodified RNase, also blocks tryptic cleavage. Thus, as noted previously by Glo¨cker et al. (24), modification at Lys41 was monitored via peptides 4061 (see below). Table 1 compares the sequence coverages obtained using different combinations of digestion method and mass spectral analytical method. In the worst case, we obtained 67% coverage of the entire RNase sequence. For simple protein identification purposes, this degree of coverage would ordinarily be considered adequate if not good. However, the observation of a single covalent modification somewhere within an entire protein de-

Chem. Res. Toxicol., Vol. 18, No. 11, 2005 1751 Table 1. Results of Analysis of RNase-(2)n by Different Methods of Digestion and MS Analysis digest MS percent method method coverage in-gel

MALDI

85

in-gel

LC/ESI

94

solution MALDI solution MALDI solution LC/ESI

67 73 100

modified lysines observed

unmodified lysines observed

1, 7, 31, 91, 98

1, 7, 31, 37, 41, 61, 91, 98 1, 7, 31, 91, 98 1, 7, 31, 37, 41, 61, 66, 98, 104 1, 7, 104 41, 61, 98 1, 7 1, 7, 31, 41, 61, 104 1, 7, 31, 37, 41, 61, 1, 7, 31, 37, 41, 61, 66, 91, 98, 104 66, 91, 98, 104

Table 2. LC/MS Analysis of Bromine-Containing Peptides Formed in Solution Digests of RNase-(2)n tR (min)

amino acid

obsd. mass (Da)a

error (ppm)b

lysine modified

17.7 38.2 42.4 37.8 32.1 67.0 69.2 63.5 42.4 33.4 32.1 60.5 63.5 69.2

1-7 1-10 2-10 11-33 34-39 38-61 40-61 40-66 62-85 86-98 92-104 99-124 1-10 (bis) 40-66 (bis)

1042.509 1474.754 1346.655 2980.294 1070.524 3208.579 2937.489 3557.813 3326.453 1876.851 1930.906 3239.651 1798.780 3881.853

49 37 60 49 56 34 51 45 30 50 46 47 35 39

K1 K1 or K7 K7 K31 K37 K41 K41 K41 or K66 K66 K91 K98 K104 K1 and K7 K41 and K61

a Each observed monoisotopic mass is the average from at least two charge states from the 79Br peptide isotope cluster. b Error is (observed - theoretical)/observed × 106.

mands either 100% sequence coverage or very good luck. The model protein RNase-(2)n contains, on average, several lysine modifications per molecule of protein. However, the peptides that we observed included only six of the 10 lysine residues in RNase, and of these, only three were modified while three others were unmodified. In a repeat of this experiment, six modified lysines were again observed, but there was only modest agreement with the first experiment in terms of which six lysines were observed and whether they were observed in modified or unmodified form. For comparison, it should be noted that proteins adducted by reactive BB metabolites in vivo often contain an average of only ca. 0.1 adduct/ molecule of protein, and for many covalent binding species, the level of adduction can be much lower (25). Furthermore, particularly in the case of BB, the adducts formed are likely to be derived from several different reactive metabolites and may be distributed among several different nucleophilic sites within a given protein molecule, thus further diluting the signal from adductbearing peptides vs others in a digest. In contrast, the best case results in Table 1 indicate that we obtained 100% sequence coverage, including two doubly modified peptides as shown in Table 2. Interestingly, among the 14 modified peptides detected by LC/ ESI-MS in the in-solution digest (Table 2), seven were relatively large with masses >2900 Da. All of these contained at least one missed cleavage, and significantly, none were observed in the in-gel digests. It is therefore not surprising that in-solution digestion followed by LC/ ESI-MS analysis resulted in a higher percent coverage of the protein sequence and allowed us to observe peptides containing all 10 lysines in both modified and unmodified forms. Perhaps the coverage by the MALDI method could have been improved by optimizing matrix

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Figure 3. Effect of increasing mass and bromine substitution on theoretical isotope patterns of model peptides. Left column, top to bottom: calculated natural abundance isotope patterns for peptides of mass 1000, 1500, 2000, and 2500. Right column, top to bottom: same as left column except for the addition of one bromine atom with a natural abundance of 79Br and 81Br. Isotope simulations were performed using a utility routine in Masslynx v. 4.0 and an assumed peptide of “polyglutamine” to reach the desired masses.

conditions and acquisition parameters, but with “real” samples of limited size, one may not have the luxury of doing this. However, for the in-gel digestion method, a more important limitation may be getting the larger peptides out of the gel for analysis. In contrast, the data in Table 2 were collected using routine chromatographic and mass spectrometric conditions with no attempt to optimize conditions for the specific sample being analyzed. Use of Isotope Signatures To Detect and Analyze Adducted Peptides. Figure 3 shows a theoretical simulation superimposing the bromine isotope pattern on that due to other isotopes commonly found in peptides (26). The spectra in the left-hand column show the natural abundance isotope patterns for peptides of mass 1000, 1500, 2000, and 2500, while those to the right show the added effect of a single bromine atom. Clearly, the isotopic signature of the bromine becomes less conspicuous at higher masses, but it is still easily recognizable up to mass 2000. For isotopic groupings with a greater

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mass spread than bromine, such as d0/d5, the signature should be quite conspicuous even at peptide masses >2000. Because each lysine modified represents a lost tryptic cleavage site, the need to be able to examine larger peptides increases; thus, the “width” of the isotopic signature becomes an important consideration in experimental design and feasibility. Actual isotope patterns of four bromine-containing peptides from among those listed in Table 2 are shown in Figure 4. These peptides have nominal masses in the range of 1000-2000, and the observed isotope patterns compare well to the theoretical patterns presented in Figure 3. The discovery of bromine-containing peptide ions that are weak or of high mass was significantly aided by comparison of spectra to the theoretical patterns in Figure 3. For most of the ions listed in Table 2, it was possible to observe the bromine isotope signature even at the very low levels of analyte eluting in the tail following their main chromatographic peak where the signal-to-noise ratio was as low as 10. To verify that the appearance of these bromine isotope patterns did not arise as an artifact from the failure of dialysis to remove residual 1 or 2 from the modified RNase preparations, we also digested unmodified RNase in the presence of unactivated 1 and 2; MS analysis of the resulting peptide mixture showed no Br-containing polypeptides (data not shown). To verify the presence of adducts and determine which lysine in peptide 1-10 (tR 38.2 min, Table 2) was modified, MS/MS analysis was performed (Figure 5). The CID spectra showed a complete series of singly charged y′′ ions from y′′1 through y′′9 and a doubly-charged y′′10 ion. The ions for y′′4 through y′′10 all contained a single bromine atom as shown by the isotope pattern, while those for y′′1 through y′′3 contained no bromine. Localization to position 7 is further confirmed by noting that ions b1-b6 show no discernible sign of a bromine isotope signature. These results clearly indicate that the modification resides on K7 and resulted in a missed cleavage at this point. Closer examination of the data also showed a weak but complete series of y′′ ions derived from a second isobaric peptide singly modified at K1 and containing a single missed cleavage at K7. Thus, although the chromatography did not separate the two isomeric peptides, they were readily distinguishable by MS/MS. The reasons for the lower abundance of the K1 modified peptide relative to its K7 isomer are that missed cleavages at unmodified lysines (i.e., K7 in the 1-10 peptide) are relatively uncommon events, so most of the RNase with a K1 modification would appear as the 1-7 peptide.

Discussion Previous workers have used proteolytic digestion and MS to evaluate the relative reactivity of individual lysine residues in RNase and have found some to be less chemically reactive than others based on observations of lesser or no modification (24, 27-29). However, our observations show that all 10 lysines in RNase were reactive toward carbodiimide-activated 2 as the electrophile. The X-ray crystal structures and NMR solution structures of RNase are nearly identical (30, 31). Inspection of these structures reveals that even the active site lysines are not very hindered. In addition, the NMR solution structure of RNase shows that all of the lysines have either no defined side chain conformation or that

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Figure 4. ESI mass spectra of peptides from in-solution tryptic digestion of RNase-(2)n. (A-D) Peptides of tR 17.7, 32.1, 38.2, and 33.4 min, respectively, in Table 2.

Figure 5. MS/MS spectrum of peptides 1-10 (KETAAAK*FER) from a tryptic digest of RNase-(2)n. The asterisk indicates modification of the lysine side chain by acylation with 2. The insets show ions b5 (KTEAA) and y′′5 (AK*FER) to illustrate the ease of recognizing the presence of bromine via its isotopic signature.

side chain motions are only moderately restricted and that all lysines have a high degree of solvent accessibility (32). Thus, it is expected that they should all be relatively reactive. Failure to observe reactions of certain lysines with acylating agents used in other studies may have been more related to the inability of the analytical method to observe all of the lysine-containing peptides, modified or unmodified, than to a fundamental lack of reactivity of certain lysines. By using RNase artificially adducted by bromophenylhistidine (1) or bromophenyllysine (2) as model proteins and submitting them to tryptic digestion and peptide mass mapping, we were able to detect all possible lysineadducted tryptic peptides containing a single “missed cleavage” simply by visual inspection of the mass spectra for the appearance of the bromine isotope signature for peptides with masses of e2000. For larger peptides, the manual inspections were facilitated greatly by comparing spectra to theoretical simulations of expected isotope clusters. Confirmation of the presence of bromine, as well as localization of the brominated residue in the peptide sequence, was achieved efficiently using modern datadependent acquisition of MS/MS spectra. Here too, the bromine isotope pattern facilitated screening of the numerous CID spectra for brominated peptides without requiring laborious interpretation of each spectrum. Bromine-containing model peptides thus behave essentially as expected upon MS analysis; they show the expected isotope clusters and are detected with sensitivity at least comparable to many ordinary tryptic peptides.

As noted in the Introduction, we have examined and identified hundreds of tryptic peptides for 45 rat liver proteins, which become adducted in vivo by metabolites of [14C]BB, and have never observed a Br-containing peptide. We believe that this is a consequence of sample heterogeneity attributable to the fact that BB gives rise to six different reactive metabolites, the major one of which (benzoquinone, 58% of total) has already lost its bromine atom. These metabolites in turn generate more than a dozen types of adducts to protein nucleophiles. With BB, the average degree of adduction of all liver proteins is 3-6 nmol equiv/mg protein (5, 14). For a 50 kDa protein, this corresponds to an average of ca. 0.150.3 adduct per molecule of protein. However, this total amount of 14C-containing adduct could potentially be subdivided among several chemical forms corresonding to multiple reactive metabolites reacting with multiple nucleophilic sites on a single protein species. As compared to the normal tryptic peptides from a radioactive protein spot on a 2D gel, the relative abundance of any individual adduct-bearing peptide could thus be quite low, making its detection very difficult even when the total sample size is completely adequate for protein identification work. Finally, if the adducted peptide is in a part of the sequence that is not normally observed in peptide mass mapping (i.e., protein identifications can sometimes be made with sequence coverages as low as 30%), it will not be seen even if it is a major adduct and it does contain bromine. Collectively, it is probably these factors, and not instumental sensitivity in the usual sense, that limits

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our ability to detect bromine-containing peptides from proteins from BB-treated rats. To obtain detectable signals from bromine-containing peptides in digests derived from “real” protein samples, it will probably be necessary to enrich the peptide mixture specifically for adducted peptides prior to MS analysis. We are currently assessing the use of immunoaffinity columns based on antibodies to haptens 1 and 2 for this purpose.

Acknowledgment. Support for this research was provided by NIH Grant GM-21784 (to R.P.H.). We thank the KU Mass Spectometry Laboratory and Dr. Tatyana Duzhak, KU Biochemical Research Service Laboratory, for technical assistance with acquiring mass spectra. The Q-TOF2 MS was purchased with support from KSTAR, Kansas administered NSF EPSCoR, and the University of Kansas. The Voyager DE-STR MS was purchased with support from NIH Grant RR13020.

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