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Anal. Chem. 1999, 71, 452-459

Characterization of Transthyretin Mutants from Serum Using Immunoprecipitation, HPLC/ Electrospray Ionization and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry R. The´berge,† L. Connors,‡ M. Skinner,‡ J. Skare,§ and C. E. Costello*,†

Mass Spectrometry Resource (R-806) and Amyloid Treatment and Research Program, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118-2526, and Pathology Department, University of Massachusetts Medical Center, Worcester, Massachusetts 01655

A mass spectrometry approach for the detection and identification of variants of the plasma protein transthyretin (TTR) is presented. The single amino acid substitutions found in TTR are closely associated with familial transthyretin amyloidosis (ATTR), a hereditary degenerative disease. A definitive diagnosis of ATTR relies on the detection and identification of TTR variants. The approach presented here is based on isolation of serum TTR using immunoprecipitation. The detection of the variant is achieved by mass measurement of the intact protein with electrospray ionization mass spectrometry (ESIMS). The liquid chromatography/ESIMS analysis of the tryptic digest of the protein followed by subsequent matrixassisted laser desorption/ionization (MALDI) time-offlight mass spectrometry and MALDI postsource decay of the relevant recovered chromatographic fraction containing the variant peptide allows the identification of unknown variants. The method was successfully tested using serum from ATTR patients with known variants (Val30fMet and Val122fIle). A new TTR variant, Ser23fAsn, was detected and identified using the above method where isoelectric focusing and restriction enzyme analysis failed to identify the nature of the variant. Familial transthyretin amyloidosis (ATTR) is a hereditary degenerative disease characterized by protein deposits which ultimately lead to organ failure(s) and death. ATTR is closely associated with single amino acid substitutions in the plasma protein transthyretin (TTR).1 The TTR monomer is constituted of 127 amino acids (molecular weight 13 761), and the protein is tetrameric in its native state. It is hypothesized that the single amino acid substitutions alter the stability of the tetramer presumably leading to aggregation or polymerization of TTR * Correspondence author: (fax) (617)-638-6491; (phone) (617)-638-6490; (e-mail) [email protected]. † Mass Spectrometry Resource, Boston University School of Medicine. ‡ Amyloid Treatment and Research Program, Boston University School of Medicine. § University of Massachusetts Medical Center. (1) Benson, M. D. The Molecular Basis of Inherited Disease, 7th ed.; McGrawHill: New York, 1995; Vol. III, pp 4159-4191.

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monomers to form amyloid fibrils.2 The clinical manifestations of ATTR are related to certain specific mutations of TTR that are typical of a particular ethnicity; e.g., Val30fMet is prevalent in Portuguese patients and Thr60fAla is known as “Irish” or Appalachian ATTR. In 1989, only 12 mutations of TTR were known. There are now over 60 known mutations3 of TTR, a minority being nonamyloidogenic. This is an indication that more TTR mutations probably exist. In the United States, the prevalence of the variant TTR genes may be as high as 1 in 100 000. The clinical profiles of ATTR may be very similar to other types of amyloidoses, but the treatments are radically different and incompatible. Hence, correct diagnosis is crucial. The reliability of the diagnosis is critical given the drastically intrusive treatment for ATTR constituted by liver transplant. In ATTR, a definitive diagnosis is established through the detection and characterization of TTR variants. The detection and characterization of established TTR variants can be achieved using isoelectric focusing (IEF) methods4 and/ or methods characteristic of molecular biology. The IEF and restriction enzyme5-based techniques are excellent and costeffective as confirmatory tests in targeted analysis. The effectiveness of screening methods in characterizing TTR variants depends on the clinical data and family history available to target the analysis. Where one is confronted with an unknown variant, the structure determination capability of mass spectrometry can be fully utilized in this clinical setting. The application of mass spectrometry to the structural analysis of protein variants has been successful, especially in cases where single amino acid substitutions are prevalent.6 The detection and characterization of hemoglobin variants represents the best example of the application of mass spectrometry to protein point mutations. Mass spectrometry has been used previously to (2) McCutchen, S.; Kelly, J. W. Biochem. Biophys. Res. Commun. 1993, 197, 415-421. (3) Benson, M. D.; Uemichi, T. Amyloid: Int. J. Exp. Clin. Invest. 1996, 3, 4456. (4) Connors, L. H.; Ericsson, T.; Skare, J.; Jones, L. A.; Lewis, D.; Skinner, M. Biochim. Biophys. Acta 1998, 1407, 185-192. (5) Nichols, W. C.; Benson, M. D Clin. Genet. 1990, 37, 44-53. (6) Wada, Y. The structure determination of aberrant proteins. In Biological Mass Spectrometry: Present and Future; Matsuo, T., Caprioli, R. M., Gross, M. L., Seyama, Y., Eds.; John Wiley and Sons: New York, 1994. 10.1021/ac980531u CCC: $18.00

© 1999 American Chemical Society Published on Web 12/05/1998

confirm the presence of a known mutation7-9 in TTR but no systematic approach to characterize an unknown has been reported. In most cases, the molecular weight difference between the wild type and variant was determined in order to confirm the proposed mutation. This method has been applied almost exclusively to the Val30fMet mutation, which is the most widespread amyloidogenic TTR mutation. Measurement of the molecular weight difference between the wild type and variant is not sufficient in itself as a general confirmatory test since other mutations may give rise to a similar mass shift. The peptide mapping of a Val30fMet variant using FABMS was reported by Wada.10 To characterize new TTR variants being discovered apace and to expedite diagnosis, the utility of various mass spectrometry approaches in analyzing ATTR patient samples is being investigated. The main purpose of the present investigation is the elaboration of a mass spectrometry-based method to detect and identify TTR variants from patient sera. Furthermore, mass spectrometry can be used to supplement current isoelectric focusing screening methods when the identity of the variant is unknown or electrophoretically “silent”. In many respects, the problem is analogous to hemoglobin variant analysis and the approaches are similar.11,12 Hemoglobin and TTR are of similar size, and both are tetrameric in their native states. A significant difference is that the blood concentration of hemoglobin is 1000-fold higher than that of TTR. In this report, we discuss the identification and characterization of TTR variants (including an unknown) using mass spectrometric techniques to demonstrate their potential in a clinical context. EXPERIMENTAL SECTION Isolation of TTR from Serum. The basic method involved isolating TTR from patient sera using immunoprecipitation.7 This was achieved by mixing an equal amount of patient serum and rabbit antiserum (Dako A/S, Glostrup, Denmark). For the molecular weight determination and tryptic digest analysis, 100 and 200 µL of serum were used, respectively. The sample was incubated overnight at 37 °C. The protein was then separated from the antibody using reversed-phase HPLC (RP-HPLC). A Vydac C4 column (300 µm, 4.6 × 250 mm) was operated at a flow rate of 0.75 mL/min (solvent A H2O/0.1%TFA, solvent B CH3CN/ 0.1%TFA). The gradient (5-60% solvent B in 40 min) was preceded with a “hold” consisting of 5 min of 95% A to ensure optimal desalting. This step yielded TTR primarily as the S-sulfated (conversion of Cys-SH to Cys-S-SO3H) and/or cysteinylated wild type and variant protein. The fractions were collected, and the solvent was evaporated using a Speed Vac. Electrospray Ionization Mass Spectrometry (ESIMS) Analysis. The S-sulfated and cysteinylated fractions were analyzed by ESIMS to detect the variant TTR on the basis of its difference in (7) Kishikawa, M.; Nakanishi, T.; Miyazaki, A.; Shimizu. A.; Nakazato, M.; Kangawa, K.; Matsuo, H. J. Mass Spectrom. 1996, 31, 112-114. (8) Ando, Y.; Ohlsson, Pl.; Surh, O.; Nylhin, N.; Yamashita, T.; Holmgren, G.; Danielsson, A.; Sandgren, O.; Uchino, M.; Ando, M. Biochem. Biophys. Res. Commun. 1996, 228, 480-483. (9) Ikeda, S.; Tokuda, T.; Nakamura, A.; Ueno, I.; Taketomi, T.; Yanagisawa, N.; Li, Y. F. Amyloid:. Int. J. Exp. Clin. Invest. 1997, 4, 104-107. (10) Wada, Y.; Matsuo, T.; Katakuse, I.; Suzuki, T.; Azuma, T.; Tsujino, S.; Kishimoto, S.; Matsuda, H.; Hayachi, A. Biochim. Biophys. Acta 1986, 873, 316-319. (11) Witkowska, E.; Shackleton, C. H. L. Anal. Chem. 1996, 68, 29A-33A. (12) Witkowska, E.; Shackleton, C. H. L. Hemoglobin 1993, 17, 227-242.

molecular weight from the wild type. A small amount (5-10 µL) of ESIMS solvent was added to the dried residue obtained from the C4 HPLC fractions. Despite the use of HPLC prior to ESIMS mass analysis, significant sodium adduction was observed. To alleviate sodium adduction by TTR, which obscured the m/z region crucial to variant detection, a small amount of cationexchange resin (ammonium form) was added to the sample solution prior to analysis. The cation-exchange resin was a BioRad AG 50W-X4 (Bio-Rad Laboratories, Richmond, CA) hydrogen form resin treated according to Nordhoff et al.13 to yield the ammonium form. The ESIMS molecular weight analysis of the collected fractions was carried out using a Micromass Quattro II mass spectrometer (Beverly, MA). The solvent was delivered into the mass spectrometer at a flow rate of 2 µL/min by a Harvard Apparatus syringe pump (South Natick, MA). The solvent consisted of a 50:50:0.1% mixture of acetonitrile, water, and formic acid, respectively. The cone voltage and capillary potential were 34 V and 3.77 kV. The ESI mass spectra were obtained over the mass range m/z 200-2000. The resulting mass spectra were obtained by summing a minimum of 20 scans. The instrument was tuned with commercially available TTR (Sigma) prior to obtaining data with the immunoprecipitated material. There was little deviation from the values of the parameters used above. The ESIMS data were mathematically deconvoluted using the MassLynx software to yield the average molecular masses and standard deviations. Treatment of immunoprecipitated TTR with DTT. Water (5 µL) was added to a sample of immunoprecipitated TTR purified by C4 RP-HPLC. A 2.5-µL aliquot was taken and was treated with 3 µL of freshly made 10 mM DTT in 30 mM NH4HCO3. The mixture was incubated at 37 °C for 1 h and subsequently drop-dialyzed14 for 15 min on a 0.025-µm pore size membrane filter (Millipore Corp., Bedford MA, Catalog No. VSWP 025 00). The solution was then mixed with an equal volume of 0.1% formic acid in acetonitrile, and the ESI mass spectrum was obtained. This mass spectrum was compared with the one obtained using the immunoprecipitated TTR not treated with DTT. Tryptic Digestion and HPLC/ESIMS. Approximately 60 µL of 0.05 M NH4HCO3 was added to the pooled cysteinylated and S-sulfated fractions obtained after HPLC of the immunoprecipitate generated from 200 µL of patient serum. Subsequently, 2-3 µL of 0.1 µg/µL TCPK-trypsin (Pierce, Rockford, IL) was added to the solution. The mixture was incubated at 37 °C for a minimum of 12 h, after which 10 µL of 10% TFA was added and the solvent was removed using a Speed Vac. A 25-µL aliquot of 2% acetonitrile/ 2% acetic acid was added to the digest, and the resulting solution was injected into the LC/MS. The separation was performed using a Vydac C18 (2.1 × 250 mm) column operating at a flow rate of 0.2 mL/min. A flow rate compatible with the mass spectrometer was obtained using a 9:1 split to the mass spectrometer. The chromatographic eluent diverted from the mass spectrometer by the split was collected at specific time windows for further matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. These time windows were selected to collect specific tryptic peptides of interest. (13) Nordhoff, E.; Ingendoh, A.; Cramer, R.; Overberg, A.; Stahl, B.; Karas, M.; Hillenkamp, F.; Crain, P. F. Rapid Commun Mass Spectrom. 1992, 6, 771776. (14) Gorisch H. Anal. Biochem. 1988, 173, 393-398.

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MALDI-TOF-MS Analysis. The recovered HPLC fractions were used directly by taking a 1-µL aliquot of the eluent and depositing the solution on 1 µL of water/2,5-dihydroxybenzoic acid (2,5-DHB) solution (10 g/L) on the MALDI probe. MALDI mass spectra were obtained with a Finnigan MAT ThermoBioAnalysis (Santa Fe, NM) Vision 2000 time-of-flight mass spectrometer equipped with a reflectron. The nitrogen laser (Laser Science Inc., Franklin, MA) had a 3-ns pulse width at 337 nm. External calibration was performed using a 10 pmol/µL mixture of ACTH clip (18-39), angiotensin, and bovine insulin. All data were obtained in the reflectron mode at 5-kV acceleration voltage with 6-kV postacceleration at the detector. The MALDI postsource decay (PSD) data were acquired in the positive ion mode with 10-kV acceleration voltage and 12.5-kV initial reflectron voltage decreased in 20% steps. RESULTS AND DISCUSSION The general strategy for the analysis of protein variants consists of two steps: detection and characterization. The mass spectrometric approach to this strategy can be broken down into three steps: (a) detection of variants through mass measurement of the intact protein molecule with electrospray ionization mass spectrometry, (b) location of the variant peptides through “peptide mapping” of the tryptic digest of the protein using LC/ESIMS or MALDI, and (c) use of MS/MS techniques to obtain sequence information from the variant peptide. This approach has been applied successfully to hemoglobin variant analysis.6,11,12 In the case of TTR, the sensitivity required is higher. This is due to the lower blood levels of TTR (0.2-0.3 g/L) compared to hemoglobin (180-200 g/L). This difference is further compounded by the diminished TTR serum levels in diseased individuals (20-50% of normal levels). An added difficulty that may present itself is the small variant to wild type ratio exhibited by certain variants. Determination of the molecular weight difference between the wild type and the variant TTR obtained by ESIMS limits postulated amino acid substitutions to a relatively small number. The next step is to analyze the tryptic digest of the protein by LC/ESIMS in order to locate more precisely the site of the mutation. Comparison of the tryptic map obtained for the variant with that of a “normal” sample facilitates detection of the modified tryptic peptide. Once the modified peptide has been pinpointed, over 90% of known mutations can be assigned at this stage. For a mutation hitherto unknown, MS/MS can be carried out on the variant tryptic peptide from the appropriate chromatographic fractions collected during the LC/ESIMS experiment. These fractions are available because of the 9:1 split to the mass spectrometer. We have the possibility to analyze such recovered chromatographic fractions using MALDI in cases where the signal-to-noise ratio of the peptide of interest is weak in the ESI mass spectrum. This situation can arise from the presence of an interfering species suppressing the ion signal or a weak ESIMS response of the peptide. The MALDI capability is an added advantage where the levels of TTR in diseased individuals are known to be reduced relative to normal levels. Another concern which can be alleviated by MALDI is the oftentimes low variant/wild type ratio which further reduces the amount of variant peptide available for analysis. Essentially, the MALDI analysis capability offers confirmatory data which can supplement the chromatographic and mass spectral data obtained in the LC/ESIMS experiment. Furthermore, se454 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999

Figure 1. ESI mass spectrum of the (a) cysteinylated and (b) S-sulfated fraction of immunoprecipitated TTR from a non-ATTR individual. * denotes species whose molecular weight is consistent with proteolytic processing of TTR.

quence information can be obtained from MALDI-PSD of the variant peptide without subsequent isolation of the peptide. Normal (Non-ATTR) Sample. The C4 RP-HPLC of the immunoprecipitate obtained from human serum generally yielded two distinctive TTR components. The ESIMS analysis of the fractions collected revealed that the two components of average molecular mass (Mr) 13 880 and 13 841 Da corresponded to cysteinylated TTR (Figure 1a) and TTR + 80 Da (Figure 1b), respectively. A small amount of unmodified TTR can also be observed (Figure 1b). Cysteinylated TTR has previously been observed in the ESIMS mass spectrum of immunoprecipitated TTR7,8 and has been detected from preparative PAGE.15 Some recent reports of electrospray mass spectrometry analysis of human TTR16,17 have assigned this TTR + 80 Da as being phosphorylated TTR. However, these reports base their assignment purely on the molecular mass increase of +80 Da from wild type TTR. A more complete characterization of the TTR + 80 Da component has recently been presented,18 and the authors concluded that this entity is in fact TTR S-sulfated at Cys10 (conversion of Cys-SH to Cys-S-SO3H). Our data are in agreement with this assignment. A crucial piece of data consisted in the disappearance of the TTR + 80 Da component from the ESI mass spectrum following incubation of immunoprecipitated TTR with (15) Petterson, T. M.; Carlstro¨m, A.; Ehrenberg, A.; Jo¨rvall, H. Biochem. Biophys. Res. Commun. 1989, 158, 341-347. (16) Green, B. N.; Oliver, R. W. A. Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, GA, May 31-June 5, 1995; ASMS: Santa Fe, NM, 1995; p 1262. (17) Hutchinson, W. L.; Alves, I. L.; Saraiva, M. J. M.; Pepys, M. B. Neuromuscular Disorders 1996, S16. (18) Kishikawa, M.; Nakanishi, T.; Miyazaki, A.; Shimizu, A. In Amyloid and Amyloidosis; Kyle, R., Gertz, M., Eds.; Parthenon Press: New York, in press.

Figure 2. UV trace (214 nm) from the LC analysis of the tryptic digest of the pooled S-sulfated and cysteinylated fractions of immunoprecipitated TTR from a non-ATTR individual. *Nonpeptidic contaminant.

the reducing agent DTT. Assignment of the TTR + 80 Da component as being TTR S-sulfated at Cys10 (the only cysteine in the TTR sequence) is the only one consistent with the effect of DTT. This assignment also explains why simultaneous cysteinylation and S-sulfation of TTR are never observed in the ESI mass spectrum of immunoprecipitated TTR since they occupy the same site. To our knowledge, there is no mention in the clinical literature of TTR existing in the S-sulfated form. In our study, the ratio of these fractions generally varied according to the source of the immunoprecipitated TTR with greater fluctuations observed in the TTR isolated from ATTR patients. Interestingly, the cysteinylated fraction was invariably more abundant in the immunoprecipitated TTR obtained from non-ATTR individuals. The S-sulfated fraction was often more abundant in diseased individuals. The significance of these observations is under investigation in our laboratory at the present time. It should be pointed out that given the number of studies investigating the effect of TTR structure on ATTR pathogenesis, the demonstration that the protein is overwhelmingly posttranslationally modified in vivo could be a significant finding. As expected, the amount of immunoprecipitated TTR (as indicated by the absorption of the TTR peak in the UV trace) isolated from the serum of non-ATTR individuals was 20-50% greater than the same serum volume from ATTR patients. LC/MS analysis of the tryptic digest of the pooled S-sulfated and cysteinylated fractions isolated from non-ATTR serum was performed. The HPLC chromatogram of the tryptic digest, shown as the UV trace in Figure 2, was very similar to those reported in the literature19,20 where incomplete cleavages for T7 and T8 as well as T11 and T12 are clearly recognizable. Most of the tryptic peptides give good ESI response and can be readily identified from their m/z values (Table 1). Even though the very hydrophilic tryptic peptides T1 and T2 elute with the solvent front and are in general not detected, this is not a great concern as only two known (19) Strahler, J. R.; Rosenblum, B. B.; Hanash, S. M. Biochem. Biophys. Res. Commun. 1987, 148, 471-477. (20) Saraiva, M. J. M.; Almeida, M. M.; Alves, I. L.; Moreira, P.; Gawinowicz, M. A.; Costa, P. P.; Rauh, S.; Banhzoff, A.; Atland, K. Am. J. Hum. Genet. 1991, 48, 1004-1008.

mutations originate from this region of TTR (Cys10fArg and Leu12fPro). Only one patient has been reported with each of these mutations. Also, the fact that no peptides having molecular weights corresponding to a cysteinylated or S-sulfated species were detected is consistent with the assignment of Cys10 as the site of these posttranslational modifications. The tryptic peptides T1 and T2 were not detected during LSIMS analysis of the unfractionated tryptic digest of TTR as reported by Wada et al.10 Val30fMet TTR Variant. The availability of data generated from the immunoprecipitated TTR originating from the serum of a non-ATTR individual allows comparison with that obtained from an ATTR patient. This comparison should quickly highlight the abnormal feature of the TTR from the diseased individual. The characterization of a known mutation from a patient having ATTR type I (Val30fMet) was successfully carried out using the above method. This was done in order to test the proposed method. The mathematically deconvoluted ESI mass spectrum of the S-sulfated fraction of the HPLC purified immunoprecipitate is shown in Figure 3. The peak (component C) corresponding to Mr 13 838.6 Da (calculated value 13 840.4 Da) is the S-sulfated wild type TTR. The peak (component D) at Mr 13 870.3 Da corresponds to the S-sulfated TTR variant. The intact TTR (component A) and its variant (component B) are also observed in the S-sulfated fraction at Mr 13 758.3 and 13 790.8 Da, respectively. The observed mass shift of +31.7 Da is consistent with a Val30fMet mutation where the expected mass shift is +32 Da. This assignment was confirmed by the results obtained with the cysteinylated fraction (data not shown). In the case of the cysteinylated fraction, the results are similar to those of Kishikawa et al.7 The main components are the cysteinylated wild type and variant TTR. The Cys-Gly (Mr 13 934 Da) and glutathione (Mr 14 061 Da) adducts of wild type TTR are also present in the ESI mass spectrum. As with many samples from ATTR patients, the S-sulfated fraction of the immunoprecipitated TTR was significantly more abundant than the cysteinylated fraction. The peptide map obtained from the UV trace recorded during the LC/MS analysis of the tryptic digest of the Val30fMet sample (Figure 4) was compared to that of a non-ATTR individual (Figure 2). Upon inspection of the chromatograms, an extra peak can be seen in the ATTR sample at a retention time of 26.76 min. This is consistent with a previously published tryptic map of a Val30fMet TTR variant where the variant peptide, T4*, appears at slightly longer retention times than T4 and elutes close to T7.21 The ESI mass spectrum corresponding to this peak yields an ion of m/z 699.86 (M + 2H)2+ which corresponds to the variant T4* (mass of 1397 Da), consistent with the expected mass shift of +32 Da. The calculated value for the (M + 2H)2+ of the variant T4* originating from the tryptic digest of the Val30fMet TTR is m/z 699.87. The mass of T4* was confirmed by MALDI analysis of the chromatographic fraction collected between retention times 25.0 and 27.9 min. The wild type and variant peptides T4 and T4* can clearly be seen in the MALDI mass spectrum, which is shown in Figure 5. The +32-Da mass shift obtained in the LC/MS and MALDI analysis of the tryptic digest is consistent with that observed in the molecular weight determination of the intact protein by ESIMS. The T4 peptide exhibits a high response to (21) Saraiva, M. J. M.; Costa, P. P.; Goodman, D. J. Clin. Invest. 1985, 76, 21712176.

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Table 1. Tryptic Peptides Resulting from the Digestion of Wild Type TTR

a

tryptic peptides

sequence

mass

T1 (1-9) T2 (10-15) T3 (16-21) T4 (22-34) T5-6 (35-48) T6 (36-48) T7 (49-70) T8 (71-76) T9 (77-80) T10 (81-103) T11-12 (104-126) T11-13 (104-127) T12-13 (105-127) T7-8 (49-76)

GPTGTGESK CPLMVK VLDAVR GSPAINVAVHVFR KAADDTWEPFASGK AADDTWEPFASGK TSESGELHGLTTEEEFVEGIYK VEIDTK SYWK ALGISPFHEHAEVVFTANDGPR RYTIAALLSPYSYSTTAIVTNPK RYTIAALLSPYSYSTTAIVTNPKE YTIAALLSPYSYSTTAIVTNPKE TSESGELHGLTTEEEFVEGIYKVEIDTK

832.4 nda 688.4 nda 671.4 1365.8 1523.6 1393.6 2455.6 703.4 582.3 2451.7 2516.9 2646.0 2489.8 3141.4

nd, not detected (elutes with solvent front).

Figure 3. Deconvoluted mass spectrum of the S-sulfated fraction of the immunoprecipitated TTR from an ATTR patient with a Val30fMet variant. A, wild type TTR mass 13 758.3 Da, B, variant TTR mass 13 790.8 Da, C, S-sulfated TTR mass 13 838.6 Da, and D, S-sulfated variant mass 13 870.3 Da.

MALD ionization and tends to be the dominant ion in the analysis of unfractionated TTR tryptic digests (data not shown). This observation perhaps could be particularly useful in devising a more sensitive and faster assay based on MALDI MS for TTR mutation detection. The method used above affords three sets of data (LC, ESIMS, MALDI MS) to determine the nature of a known mutation. Val122fIle Variant. Another example of the method consisted in analyzing an ATTR sample in which the site of the mutation is in an entirely different location from the Val30fMet example. The Val122fIle mutation is actually a polymorphism in the African-American population (4%) and might represent a hitherto neglected factor contributing to the frequency of heart disease in this population since cardiomyopathy is a major consequence of this amyloidogenic mutation.22 The ESIMS analysis of the immunoprecipitated material reveals a +14-Da mass shift consistent with Ile for Val substitution (data not shown). The peptide map obtained from the UV trace of the LC/MS analysis of the tryptic digest does not yield so straightforward an answer as in the Val30fMet example. This is due to the multiple incomplete cleavages typical of this region of TTR where T12 can (22) Jacobson, D. R.; Pastore, R.; Pool, S.; Malendowicz, S.; Kane, I.; Shivji, A.; Embury, S. H.; Ballas, S. K.; Buxbaum, J. New Engl. J. Med. 1997, 336, 466-473.

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Figure 4. UV trace (214 nm) from the LC analysis of the tryptic digest of the pooled S-sulfated and cysteinylated fractions of immunoprecipitated TTR from an ATTR patient with a Val30fMet variant.

Figure 5. MALDI mass spectrum of the chromatographic fraction collected from the LC/MS analysis of the tryptic digest of the pooled S-sulfated and cysteinylated fractions of TTR from an ATTR patient with a Val30fMet variant.

be found as T11-12, T11-13, and T12-13. The implication of these incomplete cleavages is that the signal bearing information on the T12 region of the protein is spread among four species. Furthermore, these species coelute in a relatively narrow retention time window. Hence, it is difficult to establish the presence of an “abnormal” peak upon visual inspection of the UV chromatogram. Nevertheless, abundant (M + 2H)2+ and (M + 3H)3+ ions

Figure 6. MALDI mass spectrum of the unfractionated tryptic digest of Val122Ile TTR isolated by immunoprecipitation from an ATTR patient.

corresponding to the wild type and variant tryptic peptides T11-13 and T12-13 can be detected. The location of the mutation (but not its exact position) is more easily revealed by inspection of the MALDI MS analysis of the unfractionated digest (Figure 6) where a conspicuous doublet corresponding to the wild type (m/z 2645.9) and variant T11-13 (m/z 2660.0) can be observed. Apart from T11-13 and T11-13*, T11-12 and T11-12* can also be observed at m/z 2518.4 and 2532.4, respectively. It should be stressed that the sequence coverage provided by MALDI analysis of the unfractionated digest is inconsistent and varies from sample to sample. MALDI analyses of the LC/MS fractions corresponding to the retention times of the peptides of interest yields more complete coverage. This is a further example of how MALDI and LC/ESIMS complement each other. The above techniques do not allow the differentiation of the isomeric leucine and isoleucine, which can be only be achieved by using sector instruments with high-energy CID mass spectrometry capabilities. Unknown TTR Variant. We have used the above protocol to identify an unknown TTR variant from a patient whose clinical profile was highly indicative of ATTR. The patient, a man of northern Portuguese descent, exhibited symptoms inconsistent with the mutation most commonly associated with Portuguese patients, Val30fMet. The extensive cardiomyopathy found in the subject is not a feature of Val30fMet ATTR patients. Furthermore, the currently used screening methods (isoelectric focusing and restriction enzyme analysis of most of exons 2, 3, and 4) failed to identify the nature of the mutation. The presence of a variant TTR was indicated by the observation of two strong bands in the IEF gel of the patient’s serum. Non-ATTR individuals exhibit only a single band corresponding to wild type TTR. The ESIMS analysis of the immunoprecipitated S-sulfated fraction obtained from the patient’s serum showed two distinct peaks corresponding to the wild type (Mr 13 839.0 Da, component C) and variant (Mr 13 864.9 Da, component D) S-sulfated TTR (Figure 7) as shown in the deconvoluted mass spectrum. The ESIMS results indicated a +26 ((1) Da mass shift most likely caused by single mutation. A similar mass shift was obtained for the cysteinylated fraction. The only known TTR mutation consistent with these results is Ser112fIle (+26 Da). The result of a subsequent restriction enzyme analysis targeting the Ser112fIle mutation was negative. These results, coupled with a clinical profile highly inconsistent with the ethnic origin of the patient, strongly suggested the

Figure 7. Deconvoluted mass spectrum of the S-sulfated fraction of the immunoprecipitated TTR from an ATTR patient with an unknown variant. A, wild type TTR mass 13 759.1 Da, B, variant TTR mass 13 786.5 Da, C, S-sulfated TTR mass 13 839.0 Da, D, S-sulfated variant mass 13 864.9 Da.

Figure 8. UV trace (214 nm) from the LC analysis of the tryptic digest of the pooled S-sulfated and cysteinylated fractions of immunoprecipitated TTR from an ATTR patient with an unknown variant.

existence of a new amyloidogenic TTR mutation. Hence, peptide mapping of the variant became necessary. Careful inspection of the UV chromatogram from the LC/ ESIMS analysis of the tryptic digest of the protein revealed a new peak at retention time 25.95 min (Figure 8). The ESI mass spectrum of the fraction eluting at this position showed an ion of m/z 697.29 (M + 2H)2+. The mass of this peptide (1392.6 Da) is consistent with a T4* peptide containing an amino acid substitution resulting in a +27-Da mass shift from wild type T4 (1365.7 Da). The location of the mutation on T4 was confirmed by MALDI MS analysis of the corresponding chromatographic fraction collected between retention times 25.0 and 27.5 min. In the MALDI mass spectrum, m/z 1365.2 (wild type, T4) and 1392.1 (variant, T4*) are both present (Figure 9). These results thus confirm that the mass difference between the variant and the wild type peptides is +27 Da. The only possible amino acid substitution giving rise to a +27Da shift in T4 is Ser23fAsn. To definitely assign the mutation, the MALDI-PSD spectrum of the HPLC fraction containing the wild type and variant T4 was obtained. A mass window (m/z 1350-1410) covering the molecular mass of both the wild type and variant T4 was selected for Analytical Chemistry, Vol. 71, No. 2, January 15, 1999

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Table 2. Internal Fragment Ions Observed in the PSD Spectrum of T4 and T4* mass (M + H)+

Figure 9. MALDI mass spectrum of the chromatographic fraction corresponding to retention times of 25-27.5 min and collected from the LC analysis of the tryptic digest of the pooled S-sulfated and cysteinylated fractions of immunoprecipitated TTR from an ATTR patient with an unkown variant.

Figure 10. MALDI-PSD mass spectrum of the chromatographic fraction containing the normal (m/z 1365.2) and variant T4 (m/z 1392.1) peptides collected from the LC/MS analysis of the tryptic digest of the pooled S-sulfated and cysteinylated fractions of TTR from an ATTR patient with the variant Ser23fAsn. The b and b + 27 fragment ion series are included.

the PSD experiment in order to yield a fragmentation pattern that showed both ion product series. Previous results have shown that this approach does not compromise the mass accuracy of the product ion assignment.23 The position of the modification is immediately apparent in the PSD spectrum because fragments that contain the modified amino acid exhibit doublets separated by the mass difference of the precursors whereas a common segment of the amino acid sequence would have yielded fragments of the same mass. The resulting spectrum (Figure 10) exhibited limited fragmentation in the form of a b3-6 and b3-6 + 27 ion series originating from the wild type and variant peptides, respectively. The significance of the b3-6 + 27 ion series is that the amino acid substitution resulting in the +27-Da shift must occur before or on the third amino acid in the T4* peptide. The PSD spectrum also exhibits a conspicuous ion series, all but one originating adjacent to proline and directed toward the C-terminal of the peptides. This “proline-directed internal frag(23) Perreault, H.; Horonowski, X. L.; Koul, O.; Street, J.; McCluer, R. H.; Costello, C. E. Int. J. Mass Spectrom. Ion Processes 1997 169/170, 351-370.

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internal fragment ions

observed

theoretical

PA HV and/or VH PAI PAIN PAINV PAINVA

169.4 237.5 282.6 397.7 496.3 566.7

169.1 237.1 282.2 396.2 495.3 566.3

mentation”24 is listed in Table 2 and gives further structural information. This independent set of fragment ions confirms what is indicated by the b3-6 + 27 ion series, namely, that the amino acid substitution must occur before or on the third amino acid in the T4* peptide. This incomplete fragmentation information did not provide ultimate confirmation of the nature of the mutation but supported the proposed substitution of Ser23fAsn. The mass spectrometry results were confirmed by DNA sequencing of the primer of the PCR product of the second exon. The fact that Ser23fAsn substitution required only a one base change (AGTfAAT) increases the likelihood of the mutation. It should be stressed that every TTR sample will, to some extent, present unique challenges. This expectation is due largely to the reduced TTR levels in diseased individuals as well as the different variant to wild type ratio exhibited by specific variants. Other important variables are the chromatographic properties and response to ESI and MALD ionization of the tryptic peptides containing the mutation. A fast and sensitive method based on immobilized trypsin columns and MALDI analysis could be used to supplement IEF data in cases where certain mutations are suspected but need to be confirmed. We are currently exploring the use of MALDI probes with incorporated immobilized enzymes25,26 for this purpose. Finally, the availability of clinical data and family history can also play a role in directing the analysis and reaching a definitive diagnosis. CONCLUSION We have used a combination of mass spectrometric techniques to elaborate a method of detecting and identifying variants of the plasma protein TTR isolated from patient sera. A previously unknown TTR variant (Ser23fAsn) was identified in serum from an ATTR patient. This was achieved through immunoprecipitation of TTR from patient sera, HPLC, ESIMS, and LC/MS of the tryptic digest, and MALDI MS. The mass spectrometry-based approach may not always require the complete arsenal of techniques listed above. For example, one could envision the use of MALDI-TOF MS to analyze unfractionated digests for confirmatory purposes. This type of analysis could conceivably be performed using MALDI probes incorporating immobilized enzymes. The possibility of obtaining semiquantitative variant/wild type ratios is an attractive feature of mass spectrometry analysis. (24) Spengler, B. J. Mass Spectrom. 1997, 32, 1019-1036. (25) Dogruel, D.; Williams, P.; Nelson, R. W. Anal. Chem. 1995, 67, 43434348. (26) The´berge, R.; Connors, L.; Skinner; M.; and Costello, C. E. Proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, 1998, p 159.

The mass spectrometric method described herein is not advocated as a routine screening method but rather as a highly sensitive and versatile analytical tool in situations where the clinical and screening data do not yield definitive results as to the nature of the TTR variant. Recently, we have used a combination of IEF, RFLP, and mass spectrometric methods to characterize a new TTR variant, Val122fAla.27 Given the draconian nature of present ATTR treatment (liver transplant) and the hereditary legacy of the disease, establishing a definitive diagnosis should justify the application of mass spectrometric techniques despite the comparatively high cost.

ACKNOWLEDGMENT This research was supported by NIH Grants P41 RR10888 and S10 RR10493 for the Mass Spectrometry Resource and NIH Grants R01 AR40414 and R01 AR20613 as well as the Amyloid Research Fund of the Boston University School of Medicine for the Amyloid Treatment and Research Program. The authors express special thanks to Y. Wada and M. Walsh for helpful discussions, and to Thermo BioAnalysis for the loan of the MALDI TOF mass spectrometer.

Received for review May 13, 1998. Accepted October 27, 1998. (27) The´berge, R.; Connors, L.; Skinner, M.; Skare, J.; Costello, C. E. Amyloid: Int. J. Exp. Clin. Invest., in press.

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