Anal. Chem. 2007, 79, 1990-1998
Detailed Structural Analysis of Amyloidogenic Wild-Type Transthyretin Using a Novel Purification Strategy and Mass Spectrometry Jonathan S. Kingsbury,†,‡ Roger The´berge,‡,§ John A. Karbassi,‡ Amareth Lim,‡,§ Catherine E. Costello,†,‡,§ and Lawreen Heller Connors*,†,‡
Department of Biochemistry, Amyloid Treatment and Research Program, and Center for Biological Mass Spectrometry, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118
Wild-type transthyretin (TTR), normally a soluble plasmacirculating protein, can be amyloidogenic, i.e., form tissue-deposited fibrillar material in the extracellular matrix of various organs throughout the body. Senile systemic amyloidosis (SSA) is one such pathology and features TTR-containing amyloid deposits that are found primarily in the heart. The cause for this transition from soluble to insoluble protein in SSA is yet to be determined as specific structural features that might favor TTR fibrillogenesis have not yet been identified. The precise characterization of ex vivo fibril deposits might provide insight, but structural analyses of TTR from amyloid deposits have been hindered thus far by the lack of purification strategies that overcome the insolubility of the tissue-derived protein without degrading it. Consequently, the true biochemical nature of deposited TTR remains in question. In this study, we provide detailed analyses of both the soluble (serum) and deposited (tissue) forms of TTR from cases of SSA. In the serum, a distribution of mixed disulfides, specifically S-sulfonated and S-cysteinylated forms of TTR, as well as the unmodified protein were identified. The relative levels of the three TTR species in the SSA group were comparable to amounts present in sera from age-matched control groups. For characterization of the amyloid deposited TTR, we investigated cardiac tissue samples obtained from three separate cases of SSA. We report a novel chromatographic purification strategy performed under nonreducing conditions (to maintain cysteine disulfide status) and the use of this procedure in conjunction with detailed mass spectrometric analysis of TTR from the amyloid deposits. A series of C-terminal TTR fragments with N-termini ranging from amino acids 46 to 55 were identified. We also determined that the deposits in all samples contained Cys10 disulfide-linked * Corresponding author. E-mail:
[email protected]. Tel: 617-638-4313. Fax: 617-638-4493. † Department of Biochemistry. ‡ Amyloid Treatment and Research Program. § Center for Biological Mass Spectrometry. (1) Cornwell, G. G.; Murdoch, W. L.; Kyle, R. A.; Westermark, P.; Pitka¨nen, P. Am. J. Med. 1983, 75, 618-623. (2) Pitka¨nen, P.; Westermark, P.; Cornwell, G. G. Am. J. Pathol. 1984, 117, 391-399.
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homodimers composed of full-length TTR monomers. This last finding suggests an important role for Cys10 conjugation in the transition from soluble TTR to the pathological amyloid fibril. Heart failure is a significant health issue in the aging population and, in a segment of this group, may occur as a consequence of extracellular deposits of fibrillar material known as amyloid. Autopsy studies have shown that at least 25% of all individuals over the age of 80 years have amyloid deposits in their hearts, and it is estimated that this occurrence results in fatality in up to 10% of these cases.1-3 Senile systemic amyloidosis (SSA) is a pathology associated with the deposition of amyloid fibrils in cardiac tissue and occurs predominantly in elderly men.2 The fibrils contain wild-type transthyretin (TTR)4 (Swiss-Prot accession no. P02766), a 127-amino acid (Mr 13 760.41) protein that normally circulates in plasma as a homotetramer and functions as a carrier of thyroxine and vitamin A (through association with retinol binding protein). Amyloid-deposited TTR is thought to be derived from the plasma pool of soluble TTR. Though SSA is a disease that features multiorgan involvement, the predominant clinical manifestation of SSA is cardiomyopathy. The mechanism of TTR fibrillogenesis is undetermined. Current research using recombinant proteins containing point mutations associated with familial TTR amyloidosis (ATTR) suggests that perturbation of the native tetrameric structure triggers amyloid fibril formation.5-9 This mechanism may also apply to wildtype TTR in the case of SSA. However, the identification of specific structural features that confer amyloidogenicity remains unreported. We have hypothesized that age-associated changes in thiol chemistry may play a role in the onset and progression of SSA. (3) Lie, J. T.; Hammond, P. I. Mayo Clin. Proc. 1988, 63, 552-564. (4) Westermark, P.; Sletten, K.; Johansson, B.; Cornwell, G. G. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2843-2845. (5) Lashuel, H. A.; Lai, Z.; Kelly, J. W. Biochemistry 1998, 37, 17851-17864. (6) Quintas, A.; Saraiva, M. J. M.; Brito, R. M. M. J. Biol. Chem. 1999, 274, 32943-32949. (7) Jiang, X.; Buxbaum, J. N.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14943-14948. (8) Sebastia˜o, M. P.; Lamzin, V.; Saraiva, M. J.; Damas, A. M. J. Mol. Biol. 2001, 306, 733-744. (9) Shinohara, Y.; Mizuguchi, M.; Matsubara, K.; Takeuchi, M.; Matsuura, A.; Aoki, T.; Igarashi, K.; Nagadome, H.; Terada, Y.; Kawano, K. Biochemistry 2003, 42, 15053-15060. 10.1021/ac061546s CCC: $37.00
© 2007 American Chemical Society Published on Web 01/30/2007
In our ongoing studies of serum samples from patients with ATTR and immunoglobulin light chain (primary) amyloidosis (AL), we have observed that 80-90% of circulating TTR is chemically modified at the single cysteine residue (Cys10).10-13 In both ATTR and AL, the most abundant modification is usually S-cysteinylation, but significant and sometimes even larger amounts of the S-sulfonated derivative are often present.10-13 No corresponding studies from individuals with SSA have yet been reported. Although the reason for the extensive disulfide derivatization of TTR is unclear, thiol conjugation of several proteins, including TTR, has been reported to be dependent upon age.14,15 Furthermore, biophysical studies have indicated enhanced amyloidogenicity for S-cysteinylated16 and S-sulfonated TTR,17 although there are conflicting reports on the properties of the S-sulfonated form.18 These factors together suggest a mechanism that links age and penetrance of TTR amyloidosis. In this study, we report for the first time the characterization of Cys10 disulfide status of serum circulating TTR isolated from SSA patients. In addition, we detail a novel purification strategy permitting the detailed mass spectrometric analysis of intact fibril isolates. Moreover, this is the first report to describe such analysis in TTR amyloid disease and to identify covalently linked TTR dimers in cardiac amyloid deposits. These results may represent a more accurate assessment of the structure of fibrillar TTR in SSA, at least as it occurs in the extracellular compartment of cardiac tissue. EXPERIMENTAL PROCEDURES Patients. Upon admission, patients included in this study provided a detailed history and underwent a physical examination, standardized blood tests, 24-h urine collection, chest roentogram, and verification of amyloidosis by Congo red histology of biopsy sections. A Congo red test is positive when amyloid (crossed β-sheet) fibrils are present. Patient information and biological samples were collected with institutional approval, in accordance with current IRB protocols. Autopsy tissue was obtained in accordance with the wishes of the patient or the patient’s family. For the experimental (SSA) group, a diagnosis of primary amyloidosis was excluded when histological evidence of a plasma cell dyscrasia in bone marrow biopsy sections was absent and there was no indication of a monoclonal gammopathy in the serum or urine by immunofixation electrophoresis. ATTR was ruled out if isoelectric focusing of serum19 was negative for a pathologic (10) Connors, L. H.; The´berge, R.; Skare, J.; Costello, C. E.; Falk, R. H.; Skinner, M. Amyloid 1999, 6, 114-118. (11) The´berge, R.; Connors, L. H.; Skinner, M.; Skare, J.; Costello, C. E. Anal. Chem. 1999, 71, 452-459. (12) Lim, A.; Prokaeva, T.; Connors, L. H.; Falk, R. H.; Skinner, M.; Costello, C. E. Amyloid 2002, 9, 134-140. (13) Lim, A.; Prokaeva, T.; McComb, M. E.; O’Connor, P. B.; The´berge, R.; Connors, L. H.; Skinner, M.; Costello, C. E. Anal. Chem. 2002, 74, 741751. (14) Era, S.; Kuwata, K.; Imai, H.; Nakamura, K.; Hayashi, T.; Sogami, M. Biochim. Biophys. Acta 1995, 1247, 12-16. (15) Suhr, O. B.; Svendsen, I. H.; Ohlsson, P.-I.; Lendoire, J.; Trigo, P.; Tashima, K.; Ravløv, P. J.; Ando, Y. Amyloid 1999, 6, 187-191. (16) Zhang, Q.; Kelly, J. W. Biochemistry 2005, 44, 9079-9085. (17) Kishikawa, M.; Nakanishi, T.; Miyazaki, A.; Shimizu, A. Amyloid 1999, 6, 183-186. (18) Zhang, Q.; Kelly, J. W. Biochemistry 2003, 42, 8756-8761. (19) Connors, L. H.; Ericsson, T.; Skare, J.; Jones, L. A.; Lewis, W. D.; Skinner, M. Biochim. Biophys. Acta 1998, 1407, 185-192.
TTR variant or direct DNA sequencing of the TTR gene20 showed only wild-type or nonpathogenic alleles. If the involvement of a clonal light chain and a variant TTR were both excluded, and the patient presented with amyloid cardiomyopathy at an advanced age (>60 years) without extracardiac manifestations such as renal insufficiency, nephrotic syndrome, peripheral neuropathy, orthostatic hypotension, steatorrhea, macroglossia, or purpura, the clinical diagnosis of SSA was made. The primary amyloidosis with cardiomyopathy (AL-CMP) control group consisted of patients clinically diagnosed with primary amyloidosis by the above criteria. Subjects also demonstrated cardiomyopathy, as indicated by electrocardiogram and echocardiogram, and were over 60 years of age. Subjects in the non-amyloid control group had negative Congo red biopsies, fat pad aspirates, or both and were over the age of 60 years. Reagents. Rabbit anti-human TTR antibody was purchased from DakoCytomation (Carpinteria, CA). Alkaline phosphatase conjugated anti-rabbit IgG was purchased from Sigma (St. Louis, MO). Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL). All other chemicals were from either Fisher (Fairlawn, NJ) or Sigma and were of the highest grade available. Isolation of Amyloid Components from Tissue Deposits. Amyloid fibrils were extracted from 20 g (wet weight) of autopsied heart tissue by repeated homogenization and centrifugation in 0.9% saline, 0.05 M sodium citrate in 0.01 M Tris-buffered saline, pH 8.0, and deionized water.21 The remaining pellet was divided into upper (top layer) and lower (bottom pellet) portions and lyophilized separately, as were the saline, citrate, and water washes. Either the final water wash or top layer was used for further purification. Extracted fibrils were dissolved to 10 mg/mL in 6 M guanidine hydrochloride, 0.1 M Tris, 0.01 M EDTA, pH 7.6, and the solutions were sonicated and incubated with rapid shaking overnight at 37 °C. Typically, 30 mg of extracted fibrils provided adequate soluble protein for various downstream analyses. The guanidine-soluble fibrils were rapidly exchanged into phosphate-buffered saline (PBS) (137 mM sodium chloride, 2.7 mM potassium chloride, 10.1 mM dibasic sodium phosphate, 1.8 mM dibasic potassium phosphate, pH 7.3) by two 15-min bucket dialysis steps using 10 000 molecular weight cutoff Slide-A-Lyzer cassettes (Pierce). Insoluble material was then removed by microcentrifugation at 13200×g for 5 min and clarification with 0.22-µm syringe tip filters (Nalge Co., Rochester, NY). The insoluble sedimented pellet was saved in order to test the relative solubility of the amyloid components. The clarified solution was passed through an EconoPak 10DG desalting column (Bio-Rad, Hercules, CA) preequilibrated with PBS to ensure complete dialysis. The column eluate was applied to an analytical Zorbax Poroshell 300SB-C8 HPLC (RP-HPLC) column (75 mm long, 2.1-mm i.d., 5-µm particle size) pre-equilibrated in 80% buffer A (5% acetonitrile and 0.1% trifluoroacetic acid in water), 20% buffer B (0.085% trifluoroacetic acid in acetonitrile). The sample was eluted at 1 mL/min using a linear gradient of 20-50% buffer B over 20 min with an Agilent 1100 series HPLC consisting of a G1322A degasser, a G1312A (20) Skare, J. C.; Milunsky, J. M.; Milunsky, A.; Skare, I. B.; Cohen, A. S.; Skinner, M. Clin. Genet. 1991, 39, 6-12. (21) Skinner, M.; Shirahama, T.; Cohen, A. S.; Deal, C. L. Prep. Biochem. 1982, 12, 461-476.
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pump, a G1316A thermostated column holder maintained at 70 °C, and a G1314A variable-wavelength detector set to measure absorbance at 210 nm. The fractions corresponding to the major chromatographic peaks were collected and dried in a SPD111V centrifugal concentrator (ThermoSavant, Holbrook, NY) in preparation for polyacrylamide gel electrophoresis and mass spectrometric analysis. For reduction of fibril isolates, dried fractions were resuspended in PBS containing 50 mM dithiothreitol and incubated for 1 h at room temperature. Samples were then desalted by passage through the RP-HPLC column pre-equilibrated in 95% buffer A, 5% buffer B and eluted with a 1 mL/min linear gradient of 5-85% buffer B over 5 min. Collected column eluates were dried in the centrifugal concentrator prior to downstream analyses. Isolation of TTR from Serum. TTR was immunoprecipitated from serum as described.12 The dried immunoprecipitate was dissolved in 80:10:10 (v/v/v) water/acetonitrile/acetic acid and passed through a Millipore Micron YM-100 centrifugal filter to remove the antibody. The filtrate was applied to the RP-HPLC column pre-equilibrated in 95% buffer A, 5% buffer B and eluted at 1 mL/min using a linear gradient of 5-85% buffer B over 5 min. The fractions corresponding to the major chromatographic peaks were collected and dried in the centrifugal concentrator for analysis by nanospray mass spectrometry. Tryptic Digestion. The reversed-phase purified fibril isolates (∼20-50 µg) were reconstituted in 60 µL of 0.1 M ammonium bicarbonate. Trypsin was added at 1:20 (w/w) enzyme/substrate, and the mixture was incubated at 37 °C for 15 h. SDS-PAGE and Western Blot. Purified fibril components and immunoprecipitated serum samples were dissolved in either SDSPAGE sample buffer (10 mM Tris, 1 mM EDTA, 2.5% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 0.001% (w/v) bromophenol blue, pH 8) or nonreducing SDS-PAGE sample buffer (same as above excluding 2-mercaptoethanol) and run on 10-15% gradient gels. Electrophoresis and staining with Coomassie blue or transfer to 0.45-µm nitrocellulose membranes were accomplished with a PhastSystem electrophoresis apparatus (GE Healthcare, Giles, United Kingdom) using the manufacturer’s specified procedures. Nitrocellulose membranes were probed with rabbit-anti-human TTR antibody diluted 1:1000 from 3.9 mg/mL stock in 1% dry nonfat milk in blocking buffer (150 mM sodium chloride, 10 mM Tris, 0.05% (v/v) Tween 20, pH 8) and 1:5000 (from 1.7 mg/mL stock) alkaline phosphatase conjugated anti-rabbit antibody in 5% dry nonfat milk in blocking buffer. Immunoreactive bands were visualized with BCIP/NBT Color Development Substrate (Promega, Madison, WI) according to the manufacturer’s directions. Determination of Mass Profile. Purified fibril components and immunoprecipitated serum samples were dissolved at ∼0.5 g/L in electrospray buffer (50% acetonitrile and 0.1% formic acid in water) and introduced into a Micromass Quattro II triple quadrupole mass spectrometer by nanospray using 1-µm tips prepared in-house (Sutter Instruments, Novato, CA). Continuous nanospray was generated in a 120 °C source block with the following potentials: capillary, ∼1.2 kV; cone, 32 V; extractor, 7 V; RF lens, 0.2 V. Data were obtained by scanning the range m/z 200-2000 and averaging over 1-2 min. Mass profiles (relative abundance versus mass) were produced by deconvoluting22 the (22) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 1702-1708.
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m/z data using the MAXENT function contained in the Waters MASSLYNX (v3.4) operating software. The error associated with data acquisition and processing was assumed to be (4 Da. MALDI-MS Peptide Fingerprinting. The tryptic digestions of TTR fibril isolates were neutralized with 10% trifluoroacetic acid in water and dried in a centrifugal concentrator. The samples were dissolved in 10 µL of water, and 0.5 µL of this solution was then mixed 1:1 (v/v) with water-saturated 2,5-dihydroxybenzoic acid and air-dried onto a MALDI target. Analyses were conducted using a nitrogen laser (337 nm, 3-ns pulse width) on a Reflex IV mass spectrometer equipped with a reflectron time-of-flight mass analyzer (Bruker Daltonics, Billerica, MA) and calibrated with Calibration Standard II (Bruker Daltonics). MS/MS Analysis. The tryptic digestions of TTR fibril isolates were supplemented with 65 µL of reversed-phase buffer A and filtered through 0.45-µm microcentrifuge filter devices (Corning Inc., Corning, NY). The tryptic fragments were isolated by RPHPLC using the Poroshell C8 column outlined above, eluted with a linear gradient of 5-85% buffer B over 10 min. The peak fractions were collected and dried in the centrifugal concentrator. Each fraction was resuspended in 10 µL of electrospray buffer and analyzed by nanospray using 1-µm tips prepared in-house with an Applied Biosystems QStar Pulsar i quadrupole/orthogonal acceleration time-of-flight mass spectrometer. Mass profiles for each reversed-phase peak were generated and compared to theoretical masses for TTR tryptic fragments generated by the program GPMAW (v5.02) (Lighthouse Data, Hanstholm, Denmark). These assignments were verified by mass selecting the isotopic cluster of peaks corresponding to the various tryptic fragments and analyzing each by MS/MS. Collision-induced dissociation (CID) was generated with nitrogen gas and collision energies varying from 15 to 45 eV. Analyses were also conducted on intact (undigested) RP-HPLC fractions in the same manner. Data Presentation and Statistical Analysis. Data were represented graphically using Systat SIGMAPLOT (v9.0). For evaluation of TTR immunoprecipitated from serum, the relative abundances of unmodified TTR (Mr 13761), S-sulfonated TTR (Mr 13 841), and S-cysteinylated TTR (Mr 13 881) were determined on the basis of the signal intensity of each component within the mass profile. These values were then divided by the sum of the three to determine the relative abundance of each. Relative levels of these species in SSA, non-amyloid, and AL-CMP groups were compared by one-way analysis of variance using the Analysis Tool Pack in Microsoft EXCEL. Statistical significance was assigned by the criterion of p e 0.05. RESULTS Isolation of TTR from SSA Fibrils. To directly assess the primary structure and Cys10 disulfide status of tissue-deposited TTR, a novel procedure was developed to isolate components under nonreducing conditions from cardiac-extracted amyloid fibrils. This method (see Figure 1) allowed for the first time the structural analysis of TTR amyloid fibril components using mass spectrometry. Amyloid fibrils were extracted from cardiac tissue obtained at autopsy on three individuals with SSA (cases 1-3). The fibrils were solubilized in PBS by the described methods. BCA protein assay (Pierce) indicated that ∼50% of the starting material remained soluble when exchanged into PBS. This material was
Figure 1. Strategy for isolating and analyzing fibrillar TTR from SSA cardiac amyloid deposits under nonreducing conditions.
Figure 3. Analysis of potential solubility bias in rapid buffer exchange. Pre-guanidine (lane 1), PBS-insoluble (lane 2), and PBSsoluble (lane 3) fractions of a cardiac fibril extraction from case 1 were analyzed by nonreducing 10-15% SDS-PAGE to determine whether the purification method introduced a bias due to differential solubility of amyloid components. Total protein was detected by Coomassie blue staining (left panel) and TTR was detected by antiTTR Western blot (right panel). Apparent molecular weight positions were established using standard markers (GE Healthcare), as indicated. Also indicated are positions corresponding to commercial TTR (Sigma). Figure 2. RP-HPLC chromatograms of extracted cardiac fibrils from three cases of SSA. PBS-soluble amyloid fractions from each case were applied to an analytical Zorbax Poroshell 300SB-C8 HPLC column pre-equilibrated in 20% buffer B and eluted over 20 min with a 20-50% linear increase in buffer B. Arrows indicate the major peaks that were collected and screened for TTR by SDS-PAGE/Western blot analysis and nanospray mass spectrometry. The two TTRcontaining isolations (bordered by dashed lines) were termed the early (9) and late (b) fractions.
then purified by RP-HPLC as indicated in Figure 2. The fractions of eluent corresponding to several prominent features on the three chromatograms (indicated with arrows in Figure 2) were collected and screened by SDS-PAGE and Western blot (data not shown), as well as by nanospray mass spectrometry, to identify fractions containing TTR-related components. Of the indicated fractions, two contained species that were consistent with truncated or fulllength TTR by our initial screening. The peak at the retention time of ∼5.75 min (indicated by 9 in Figure 2) did not react with the anti-human TTR antibody used in this study, but ran at a position on the gel consistent with previous reports of truncated TTR.4,23,24 Nanospray mass spectrometry of this fraction further supported this assignment (see below). The peak at the retention time of ∼10.75 min (indicated by b in Figure 2) was immunore(23) Gustavsson, Å.; Jahr, H.; Tobiassen, R.; Jacobson, D. R.; Sletten, K.; Westermark, P. Lab. Invest. 1995, 73, 703-708. (24) Bergstro¨m, J.; Gustavsson, Å.; Hellman, U.; Sletten, K.; Murphy, C. L.; Weiss, D. T.; Solomon, A.; Olofsson, B.-O.; Westermark, P. J. Pathol. 2005, 206, 224-232.
active. These two peaks from each of the three cases were selected (as indicated by the dashed borders in Figure 2) for further analysis and termed “early peak” (9) and “late peak” (b), respectively. To examine the possibility that the relative solubility of fibril components in guanidine and PBS might bias the interpretation of the downstream analyses, SDS-PAGE and Western blot analyses were used to obtain comparative assays of the component distribution of the starting material (tissue-extracted fibrils), as well as the PBS-insoluble and soluble fractions (Figure 3). These analyses indicated a similar distribution of components in all the samples, suggesting that our method was not selective with respect to solubility. Characterization of TTR Fragments from SSA Fibrils. Mass profiles for the early peak RP-HPLC fractions were generated by electrospray ionization mass spectrometry and are shown in Figure 4. The profiles for all cases were similar and consisted of a discrete range of average neutral masses between 8178 and 9040 as indicated in Table 1. Calculations based on the amino acid sequence of TTR suggested that these species correspond to a series of C-terminal fragments with N-termini ranging from amino acids 46-55 (except 54). Our assignments include all the TTR truncations that have been previously identified by Edman sequencing4,23,24 as well as additional truncations. The intervals between the peaks each correspond to the mass of a single amino acid residue (except species #), providing a sequence tag readily determined by mass spectrometry. Analytical Chemistry, Vol. 79, No. 5, March 1, 2007
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Table 1. Species Present in the Early-Peak RP-HPLC Fractionsa observed neutral mass peak
case 1
case 2
case 3
TTR sequence assignment
theoretical mass (av)
A B C D E F G H I
8178
8178
8450
8451
8667 8768 8896 8953 9040
8666 8768 8896 8953 9040
8176 8364 8450 8580 8666 8767 8895 8953 9040
Leu55-Glu127 Gly53-Glu127 Ser52-Glu127 Glu51-Glu127 Ser50-Glu127 Thr49-Glu127 Lys48-Glu127 Gly47-Glu127 Ser46-Glu127
8177.13 8363.30 8450.37 8579.49 8666.57 8767.67 8895.85 8952.90 9039.98
a Letters A-I correspond to peak assignments in Figure 4. TTR sequence assignments that represent newly identified species are in italic type.
Figure 4. Nanospray mass spectrometry of the early-peak RPHPLC fractions. Early-peak RP-HPLC fractions from each SSA case were resuspended in electrospray buffer and introduced into a Micromass Quattro II triple quadrupole mass spectrometer by nanospray. The raw data (m/z) were deconvoluted with the MAXENT function contained in the operating program MASSLYNX (v3.4). Observed neutral masses are shown in the inset.
We confirmed these assignments by tryptic digestion of each fraction followed by MALDI-MS peptide fingerprinting and MS/ MS of the digestion products. These analyses provided several insights into the composition of the early-peak RP-HPLC fractions. First, although these fractions were not recognized by the antihuman TTR antibody employed in this study, we confirmed the presence of several prominent TTR tryptic fragments (Val71Lys80, Ala81-Arg103, Arg104-Lys126, Tyr105-Lys126, Arg104Glu127) by MALDI-MS peptide fingerprinting, thus proving that the species were indeed portions of the TTR molecule. Second, we conducted a more detailed analysis of the tryptic fragments from case 2 using MS/MS. In this analysis, we confirmed the above fragment assignments. We also observed and analyzed several ions that were attributed to portions of the TTR sequence not observed in the nanospray mass profile (Figure 4). These TTR isoforms may be less obvious in the profile because they had low abundances relative to the main fragments observed and listed in Table 1. One of these was an [M + 2H]2+ ion at m/z 683.90, consistent with the Gly22-Arg34 tryptic fragment of TTR (theoretical [M + 2H]2+ m/z 683.88). We also detected an [M + 3H]3+ ion at m/z 408.26. MS/MS analysis identified this as a truncated version of the above tryptic fragment, corresponding to residues Pro24-Arg34. Since trypsin would not cleave the Ser23-Pro24 peptide bond, observation of this species suggests the presence of a minor N-terminally truncated species not included in the sequence tag (assuming specificity of the commercial trypsin). In addition, we observed two [M + 2H]2+ ions at m/z 759.47 and 1994 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007
681.44. MS/MS analysis identified these as partial tryptic fragments corresponding to residues Arg104-Tyr116 and Tyr105Tyr116 respectively, suggesting that a C-terminal truncation at Tyr116 was also present in our fibril preparation. In addition to these ions, we also observed an [M + 3H]3+ ion at m/z 713.72. This was identified by MS/MS as a partial tryptic fragment corresponding to residues Ser52-Lys70, consistent with the assignment of the Mr 8450 species as an N-terminal truncation at position 52 (see Table 1). Detection of this ion supported the sequence tag established by the mass profile (see Figure 4). However, since TTR is cleaved by trypsin between Lys48 and Thr49, the most abundant TTR truncated species could not be verified by these analyses, as the N-termini of the fragments would generate tryptic peptides too small to be easily detected. Therefore, the sequences of the fragments were established by direct MS/MS (without tryptic digestion), which confirmed the assignments shown in Table 1. “Top-down” MS/MS analysis25,26 was conducted on four ions isolated from the early-peak RP-HPLC fraction of case 3. The ions of m/z 904.96 [M + 10H]10+, 975.19 [M + 9H]9+, 939.89 [M + 9H]9+, and 909.60 [M + 9H]9+ were mass selected for MS/MS analysis as they were assigned to the C-terminal TTR fragments having neutral masses of m/z 9040, 8768, 8450, and 8178, respectively. MS/MS analysis of these ions generated fragments corresponding to an intact C-terminus (ions consistent with TTR y1+-y9+ were observed). Furthermore, a series of b fragment ions originating via cleavages at positions 61-64 were also identified. These findings unequivocally described the N-terminus of the selected species, providing further evidence to support the assignments listed in Table 1. An example of these data is shown in Figure 5. These results provide the first identification of this series of truncated TTR species under nonreducing and undigested (i.e., top-down) conditions. Top-down MS/MS analysis was also used to characterize a Mr 8723 species that was present in the mass profile (labeled # in Figure 4) of all three cases, but does not represent a simple truncation of the TTR sequence. The observed neutral mass of this species was between that of species E (Mr 8666) and F (Mr (25) Kelleher, N. L.; Lin, H. Y.; Valasokovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806-812. (26) Kassai, H.; Satomi, Y.; Fukada, Y.; T’akao, T. Rapid Commun. Mass Spectrom. 2005, 19, 269-274.
Figure 5. MS/MS analysis of the peak at m/z 975.19 [M + 9H]9+. The early-peak RP-HPLC fraction from the case 3 fibril purification was resuspended in electrospray buffer and introduced into an Applied Biosystems QStar Pulsar i quadrupole/orthogonal acceleration timeof-flight mass spectrometer. The isotopic cluster of the peak at m/z 975.19 [M + 9H]9+ (corresponding to the residue 49-127 truncated species) was selected and fragmented in the instrument’s collision cell. Assignments of the observed y-series product ions to the C-terminal sequence of TTR are indicated. A peak at m/z 148.06 with an intensity of 2.71 corresponds to the y+ ion but occurs in a very heterogeneous region of the spectrum. The series of three b2+ ions could be assigned to b142+, b152+, and b162+, as indicated.
8767), suggesting a chemically modified form of one of these fragments (Mr 8666 + 57 or Mr 8767 - 44). To identify the species, top-down MS/MS was conducted on ions of m/z 873.2 [M + 10H]10+, 970.2 [M + 9H]9+, and 1091.2 [M + 8H]8+ (the three most abundant charge states of Mr 8723 that were observed) from the early-peak RP-HPLC fraction of case 2. Three different charge states were selected and analyzed in order to maximize the sequence coverage, as variations in fractionation efficiency are possible. Analysis of each of the three ions provided similar results and indicated that the species in question contains the native intact TTR C-terminus (ions consistent with TTR y1+-y9+ were observed). Furthermore, b fragment ions originating via CID-induced cleavages at positions 59-64 were observed. The masses of this ion series followed the native TTR sequence but included the mass shift observed for the molecular ion, indicating that the modification occurs on one of the first 10 amino acids of the truncated N-terminus. The average difference between the [M + 7H]7+, [M + 9H]9+, and [M + 10H]10+ charge states of the chemically modified species and species F was 43.9998 Da. The calculated monoisotopic mass of CO2 is 43.9898 Da, strongly suggesting that the species in question is a decarboxylated form of species F. Characterization of Full-Length TTR from SSA Fibrils. Mass profiles for the late-peak RP-HPLC fractions were generated by nanospray mass spectrometry and are shown in Figure 6. Two of the three cases contained a predominant species with an average neutral mass of ∼27 521 and minor quantities of a species of Mr 13 761. The preparation from case 3 was heterogeneous, and the individual molecular ions were not resolved from one another within the broad peak centered around m/z 27 500. It is not unexpected to observe variation in the biochemical purification efficiency between samples, as extracted amyloid fibrils are heterogeneous mixtures with varying composition between cases (as illustrated by the varying mass profiles in Figure 4). However, despite the lack of a mass profile for case 3, the presence of a species consistent with a TTR dimer was confirmed by Western blot (see below). In the two mass profiles, the measured neutral masses correlate closely with the expected mass of the TTR dimer
Figure 6. Nanospray mass spectrometry of the late-peak RP-HPLC fractions. Late-peak RP-HPLC fractions from each SSA case were resuspended in electrospray buffer and introduced into a Micromass Quattro II triple quadrupole mass spectrometer by nanospray. The raw data (m/z) were deconvoluted with the MAXENT function contained in the operating program MASSLYNX (v3.4). The late-peak RP-HPLC fraction from case 3 was too heterogeneous to generate a definitive mass profile.
Figure 7. Nonreducing and reducing Western blot analysis of the late-peak RP-HPLC fractions. Late-peak RP-HPLC fractions from each SSA case (indicated by the lane numbers) along with a TTR control from pooled human serum (Sigma) were run under nonreducing and reducing conditions on a 10-15% gradient SDS-PAGE gel. The gel was visualized by Western blot using an anti-human TTR antibody (DakoCytomation).
(27 518.82) and monomer (13 760.41), respectively. Since the major species had a mass consistent with TTR dimer and noncovalent interactions are expected to be disrupted under the nanospray conditions employed here, we hypothesized that the TTR in these fractions consisted predominantly of monomers that were cross-linked by disulfide bonds at Cys10, the only Cys in wild-type TTR. This possibility was further investigated by performing nonreducing and reducing Western blot analyses on the late-peak RPHPLC fractions. As indicated in Figure 7, a predominant band consistent with the dimer in the control TTR lane was present in all three cases under nonreducing conditions. In each case, reduction of the samples with 2-mercaptoethanol resulted in the appearance of a band at a position consistent with TTR monomer. These results, in conjunction with the nanospray mass profiles, strongly suggested that the TTR in the late-peak RP-HPLC fractions contained monomers that were linked by disulfide bonds at Cys10. The presence of significant residual band density at the dimer position in the reduced samples was attributed to SDS-resistant, Analytical Chemistry, Vol. 79, No. 5, March 1, 2007
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Table 2. Descriptive Statistics and Analysis of Variance of the Distribution of TTR Cys10 Mixed Disulfides in SSA, AL-CMP, and NA Seruma
n M/F age %TTRsulf %TTRcys %TTRunmod
SSA
AL-CMP
NA
p-value
20 20/0 73 ( 1.39 32.90 ( 3.36 56.20 ( 2.90 10.90 ( 1.12
20 13/7 71 ( 1.27 30.95 ( 3.88 57.40 ( 4.22 11.65 ( 1.35
10 4/6 70 ( 2.62 32.60 ( 7.77 55.80 ( 8.06 11.60 ( 1.14
0.939 0.967 0.889
a Variables are listed as mean ( standard error of the mean. Statistically significant differences were assigned as p e 0.05. No female patients were included in the SSA group since symptomatic disease is found predominantly in men (2). There were no distinctive sexassociated differences between results for the samples from the males (M) and females (F) among the AL and control patients.
Figure 8. Nanospray mass spectrometry of unreduced and reduced (+DTT) late-peak RP-HPLC fractions from case 1. The late-peak RPHPLC fraction from SSA case 1 was treated with 50 mM DTT in PBS for 1 h, desalted by RP-HPLC, and dried in a centrifugal concentrator. The pellet was resuspended in electrospray buffer and introduced into a Micromass Quattro II triple quadrupole mass spectrometer by nanospray. The deconvoluted mass spectrum obtained after reduction was then compared to the deconvoluted spectrum obtained for the unreduced sample. The peaks assigned to the major TTR species (dimer in the unreduced sample and monomer in reduced sample) are indicated in boldface type. Minor species are indicated in normal type.
noncovalent rather than covalent interactions, since a band at this position is always apparent in reducing SDS-PAGE analysis of any TTR sample especially when visualized by Western blot (see control lane). However, to unequivocally establish the presence of intersubunit cystine, the late-peak RP-HPLC fraction from case 1 was reduced with dithiothreitol. The nanospray mass spectrum obtained after reduction was compared to the spectrum of the unreduced sample. As shown in Figure 8, there was a shift in the predominant species from an average mass of 27 521 to 13 761, consistent with a dithiothreitol-induced conversion from dimer to monomer. In both the unreduced and reduced samples, heterogeneity of the TTR isoforms was detected. For example, the minor peak at mass 27 367 ( ∼155 Da lower than that of the major peak) likely corresponds to a disulfide-linked TTR dimer that consists of one full-length monomer and one truncated monomer that lacks the N-terminal Gly-Pro residues. This assignment is consistent with the appearance of the Mr 13 606 species upon reduction, which closely approximates the mass of the truncated monomer (theoretical Mr 13 606.24). An additional peak at mass 13 913 in the reduced sample may correspond to a non-disulfide-linked glutathione adduct of this truncated species (+307 Da). Overall, these results furnish the first evidence that disulfide-linked TTR subunits are a predominant component of cardiac amyloid deposits in individuals with SSA. Distribution of TTR Mixed Disulfides in Serum. Mass profiles of TTR immunoprecipitates from 20 SSA, 20 immunoglobulin light chain amyloidosis with cardiomyopathy (AL-CMP), and 10 non-amyloidosis (NA) serum samples were generated by nanospray mass spectrometry. Results were compared by analysis of variance with respect to the relative amounts of TTR in the S-sulfonated (%TTRsulf), S-cysteinylated (%TTRcys), and unmodified 1996 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007
(%TTRunmod) forms. Descriptive statistics for the three groups and the results of this analysis are presented in Table 2. One-way analysis of variance did not detect significant differences in these variables. Disulfide-linked TTR dimers were not observed in any of the TTR serum immunoprecipitates. DISCUSSION The data presented in this report provide a detailed analysis of the primary structure and cysteine modification of TTR circulating in plasma and deposited as amyloid fibrils in cardiac tissue from individuals with SSA. Detailed structural analyses of fibrillar TTR have been thus far hindered by the lack of procedures that provide the purity required for mass spectrometry. Here, we have reported a novel, nonreducing purification strategy for isolating TTR amyloid components from tissue. This procedure enabled the first detailed structural characterization of fibrillar TTR by mass spectrometry. In addition, since this procedure is nonreducing, the disulfide status of the isolated components has been maintained. We have also reported the use of existing methods10-13 to isolate and analyze the primary structure and disulfide status of serum-circulating TTR in individuals with SSA. We have reported for the first time, the relative abundance of the three predominant forms of circulating TTR (S-sulfonated, S-cysteinylated, unmodified) in an SSA population. SSA Cardiac Deposits Contain Abundant C-Terminal TTR Fragments. We have identified abundant C-terminal TTR fragments in cardiac amyloid deposits from three cases of SSA. These data confirm the presence and extend the characterization of TTR forms included in previous reports.4,23,24 In addition, these results validate our method of purification as consistent with other methods, while providing the level of purity required for detailed mass spectrometric analyses. Interestingly, no corresponding N-terminal fragments were identified, a finding also reported with other purification methods.4,23,24 This observation may indicate that the endoproteases responsible for N-terminal truncation remove individual amino acids or short peptides that would be quite volatile and not be detected in either the nanospray or MALDI mass spectra. Our approach has allowed, for the first time, the analysis of tissue-deposited TTR fragments by mass spectrometry and contributes two important features not available by previously employed methods. First, the sensitivity and specificity of this technique allowed the identification of truncation sites that had
not, to our knowledge, been conclusively identified (fragments starting at positions Gly47, Lys48, Ser50, Glu51, and Leu55 are novel; fragments starting at positions Ser46, Thr49, Ser52, and Gly53 have been previously reported). The results presented herein identify a complete range of cleavages from positions 46 to 55 with the exception of position 54. Cleavage between Gly53 and Glu54 would result in a Mr 8306.24 fragment. The reason for the absence of this component is not immediately obvious but may indicate endoprotease specificity. Second, in addition to increasing the sensitivity, mass spectrometric analysis of the amyloid fibril deposits allowed the relative abundances of all species to be semiquantitatively determined from a single data set. For instance, case 1 and case 3 both share the 46-127 fragment as the most abundant, while in case 2, it is the 49-127 species. More analyses will be required to establish whether such variations in the fragment profile are important in the disease process. SSA Cardiac Deposits Contain Abundant Cys10 DisulfideLinked TTR Subunits. In addition to C-terminal TTR fragments, we have also purified and characterized full-length TTR from amyloid deposits in the same three SSA autopsied hearts. Of the full-length TTR fractions, disulfide-linked dimer was the predominant form, with minor quantities of unconjugated monomer (undetected by Western blot and representing only 10-15% of the dimer signal by nanospray mass spectrometry). No Ssulfonated or S-cysteinylated TTR was identified, though our results do not eliminate the possibility that these species are present at low levels in SSA fibrils. These findings suggest that the distribution of TTR chemical species in amyloid deposits differs significantly from that in serum. This report is the first to conclusively identify Cys10-Cys10 disulfide-linked TTR in cardiac amyloid deposits from cases of SSA. Though this species was implicated over a decade ago in vitreous amyloid from several ATTR (Val30Met) patients by SDSPAGE,27 the suggestion was only speculative at that time, since isolation and structural characterization of the proposed species were not reported. The results reported herein support the interpretation of the SDS-PAGE data from Thyle´n et al.27 and also suggest that Cys10-Cys10 disulfide linkages are formed in wildtype (SSA) TTR deposits in addition to Val30Met (ATTR) TTR deposits. The origin, function, and pathological importance of the disulfide bond formation between monomers in the TTR amyloidoses are unclear, but this study indicates that the modification is abundant in cardiac deposits and suggests that further studies are warranted. Based on crystallographic data using recombinant TTR (Val30Met), a model for amyloid formation that includes the formation of disulfide bonds has been proposed,28 although it has (27) Thyle´n, C.; Wahlqvist, J.; Haettner, E.; Sandgren, O.; Holmgren, G.; Lundgren, E. EMBO J. 1993, 12, 743-748. (28) Terry, C. J.; Damas, A. M.; Oliveira, P.; Saraiva, M. J. M.; Alves, I. L.; Costa, P. P.; Matias, P. M.; Sakaki, Y.; Blake, C. C. F. EMBO J. 1993, 12, 735741. (29) McCutchen, S. L.; Kelly, J. W. Biochem. Biophys. Res. Commun. 1993, 197, 415-421. (30) Karlsson, A.; Olofsson, A.; Eneqvist, T.; Sauer-Eriksson, A. E. Biochemistry 2005, 44, 13063-13070. (31) Nelsestuen, G. L.; Zhang, Y.; Martinez, M. B.; Key, N. S.; Jilma, B.; Verneris, M.; Sinaiko, A.; Kasthuri, R. S. Proteomics 2005, 5, 4012-4024. (32) Nedelkov, D.; Kiernan, U. A.; Niederkofler, E. E.; Tubbs, K. A.; Nelson, R. W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10852-10857.
been established that disulfide formation is not required for acidmediated fibrillogenesis.29 The presence of dimeric TTR in amyloid deposits may indicate that monomer cross-linking through disulfide bond formation is important in the mechanism of fibril formation, specifically in TTR-associated forms of amyloid disease, i.e., SSA and ATTR. Our data do not provide temporal information about disulfide bond formation, i.e., whether the bond forms before or after amyloid fibril assembly. However, a recent report30 has demonstrated that a recombinant TTR double mutant, Cys10Ala/Tyr114Cys, when dimerized by a disulfide bond through the introduced cysteine, can form amyloid protofibrils that are morphologically similar to those formed under reducing conditions. The study does not directly examine the amyloidogenicity of the covalently linked dimer relative to the non-disulfide form. However, the ability of the Cys114-Cys114 disulfide-linked dimer to form amyloid does suggest that it is possible for Cys10-Cys10 disulfide formation to precede fibrillogenesis, though this possibility has yet to be examined experimentally. In contrast to the Cys10-Cys10 disulfide forming prior to fibrillogenesis, it is also possible that this bond could occur after fibrilization and enhance the stability of the deposit. Lack of high-resolution structural information of ex vivo fibril deposits renders this possibility difficult to assess. For this purpose, studies of appropriately modified recombinant TTR may prove useful in determining how Cys10Cys10 disulfide formation contributes to amyloid fibrillogenesis and stability. Cys10 Conjugation of TTR Purified from the Serum of Patients with SSA Consists Mainly of S-Sulfonated and S-Cysteinylated Isoforms. At present, the diagnosis of SSA is based on several criteria including the exclusion of other more common forms of amyloidosis with overlapping clinical symptoms. In conjunction with biopsy proof of amyloid, the detection of a structural feature unique to SSA would facilitate the differential diagnosis. TTR mixed disulfides have recently been suggested as possible biomarkers for various conditions.31,32 We characterized all major forms of TTR present in serum samples from cases of SSA and further investigated whether the proportions of circulating S-sulfonated and S-cysteinylated TTR in this population might differ from other age-matched non-SSA samples. Comparison of sera from cases of SSA (n ) 20), AL-CMP (n ) 20), and non-amyloid controls (n ) 10) indicated that the profile of TTR chemical species was, on average, comparable among the groups. While these serum results in the SSA group do not provide evidence to confirm early indications that the S-sulfonation/S-cysteinylation ratio might serve as a diagnostic marker, there are two considerations for the interpretation of these findings. First, the number of subjects is relatively small, limiting the power of the study. Second, though age was controlled, it is difficult to account for all confounding variables, particularly in this age bracket where various undiagnosed health problems could complicate the interpretation of the data. The results reported herein do affirm that our caution against overinterpretation of the early results was justified and strengthens the argument that careful analysis is required to follow up on preliminary observations. CONCLUSIONS A novel method for purifying components from tissue-deposited amyloid fibrils in the absence of reductants has been developed Analytical Chemistry, Vol. 79, No. 5, March 1, 2007
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and represents a significant improvement over previously reported strategies. This approach permitted the structural assignment of the major TTR-related components in SSA-deposited fibrils found in human heart tissue. These assignments included a series of N-terminally truncated species as well as full-length disulfide-linked dimer, a previously unreported component. The disulfide status of serum-circulating TTR has been investigated in SSA patients and control samples and found to be similar among the groups, but to vary from the TTR forms identified in amyloid deposits from the cardiac specimens. ACKNOWLEDGMENT The authors thank Dr. Martha Skinner for helpful discussions and for critical review of the manuscript. Support for this study
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was provided by grants from the American Heart Association (AHA0060149T to L.H.C.), the National Institutes of Health (P41 RR10888, S10 RR10493, and S10 RR15942 to C.E.C.), the Gerry Foundation, the Young Family Amyloid Research Fund, the Eileen Cochran Amyloid Research Fund, and the David S. Levine Amyloid Research Fund. J.S.K. and R.T. contributed equally to this work.
Received for review August 18, 2006. Accepted December 19, 2006. AC061546S
