Characterization of Transthyretin Variants in ... - ACS Publications

Application to familial amyloidotic polyneuropathy type I. Laura Pont , Fernando Benavente , José Barbosa , Victoria Sanz-Nebot. ELECTROPHORESIS 2015...
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Anal. Chem. 2002, 74, 741-751

Characterization of Transthyretin Variants in Familial Transthyretin Amyloidosis by Mass Spectrometric Peptide Mapping and DNA Sequence Analysis Amareth Lim,†,‡,§ Tatiana Prokaeva,§,| Mark E. McComb,†,‡ Peter B. O’Connor,†,‡,§ Roger The´berge,†,‡,§,⊥ Lawreen H. Connors,‡,§ Martha Skinner,§,| and Catherine E. Costello*,†,‡,§,#

Mass Spectrometry Resource, Departments of Biochemistry, Medicine, Physiology and Biophysics, and Amyloid Treatment and Research Program, Boston University School of Medicine, Boston, Massachusetts 02118

Transthyretin (TTR) is a 127-amino acid residue transport protein. In plasma, TTR exists as a tetramer and binds the hormone thyroxine and the retinol-binding proteinvitamin A complex. Amino acid substitutions in TTR are hypothesized to destabilize the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with extracellular deposition of wild-type TTR, its variants or fragments as amyloid fibrils in various tissues and organs. A definitive diagnosis of ATTR depends on the detection and identification of TTR variants. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), in combination with trypsin digestion, have been shown to be powerful tools in characterizing TTR variants. Typically, TTR or its tryptic digest is analyzed by MALDI-TOF MS, liquid chromatography ESI MS, or both. Analysis of tryptic digests by MALDI-TOF MS does not provide enough sequence coverage in TTR to identify all possible modifications. To improve sequence coverage, aliquots of immunoprecipitated TTR samples were digested with trypsin, lysyl endopeptidase Lys-C, or endoproteinase Asp-N. Identification of the peptides from each digest by MALDI-TOF MS provided preliminary information about the sites and mass shifts due to amino acid substitutions from genetic mutations and to posttranslational modifications. The location and identity of the modifications in the variant proteins were then confirmed by tandem mass spectrometry, accurate mass measurements, and direct DNA sequence analysis. Using these methodologies, we achieved 100% sequence coverage. The detection of two nonpathologic variants (Thr119Met and Gly6Ser) and four pathologic variants (Phe64Leu, Asp38Ala, Phe44Ser, and previously unreported Trp41Leu) are described as illustrations of this approach. Transthyretin (TTR, formerly called prealbumin) is a homotetramer with each subunit consisting of 127 amino acid residues. Although it is synthesized predominantly in the liver and secreted into plasma, TTR is also produced in the choroid plexus and the eye. In plasma, the tetrameric TTR transports the hormone 10.1021/ac010780+ CCC: $22.00 Published on Web 01/15/2002

© 2002 American Chemical Society

thyroxine and the retinol-binding protein-vitamin A complex.1 TTR is associated with senile systemic amyloidosis (SSA) and familial transthyretin amyloidosis (ATTR).2-5 SSA, also referred to as senile cardiac amyloidosis (SCA), is a nonhereditary disorder that affects about 25% of individuals over 80 years old.6 In SSA, amyloid fibrils are usually composed of wild type (wt) TTR, its fragments, or both and are found mainly in the heart.7-9 In vitro studies suggest that S-sulfation of wt TTR is highly amyloidogenic and possibly plays an important role in SSA.10 In contrast, ATTR is an autosomal dominant disorder involving the deposition of TTR variants, the wt protein, or their fragments as amyloid fibrils in various tissues and organs.11-13 Although the incidence of ATTR is unknown, the gene frequency for TTR * To whom correspondence should be addressed: (telephone) (617) 6386490; (telefax) (617) 638-6491; (e-mail) [email protected]. † Mass Spectrometry Resource, Boston University School of Medicine. ‡ Department of Biochemistry, Boston University School of Medicine. § Amyloid Treatment and Research Program, Boston University School of Medicine. | Department of Medicine, Boston University School of Medicine. ⊥ Present address: Genzyme Corp., 1 Mountain Rd., Box 9322, Framingham, MA 01701-9322. # Department of Physiology and Biophysics, Boston University School of Medicine. (1) Rostom, A. A.; Sunde, M.; Richardson, S. J.; Schreiber, G.; Jarvis, S.; Bateman, R.; Dobson, C. M.; Robinson, C. V. Proteins 1998, (Suppl. 2), 3-11. (2) Benson, M. D. The Metabolic and Molecular Bases of Inherited Disease; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; pp 4159-4191. (3) Falk, R. H.; Comenzo, R. L.; Skinner, M. N. Engl. J. Med. 1997, 337, 898909. (4) Damas, A. M.; Saraiva, M. J. J. Struct. Biol. 2000, 130, 290-299. (5) Hund, E.; Linke, R. P.; Willig, F.; Grau, A. Neurology 2001, 56, 431-435. (6) Cornwell, G. G.; Murdoch, W. L.; Kyle, R. A.; Westermark, P.; Pitka¨nen, P. Am. J. Med. 1983, 75, 618-623. (7) Westermark, P.; Sletten, K.; Johansson, B.; Cornwell, G. G. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2843-2845. (8) Christmanson, L.; Betsholtz, C.; Gustavsson, Å.; Johansson, B.; Sletten, K.; Westermark, P. FEBS Lett. 1991, 281, 177-180. (9) Gustavsson, Å.; Jahr, H.; Tobiassen, R.; Jacobson, D. R.; Sletten, K.; Westermark, P. Lab. Invest. 1995, 73, 703-708. (10) Kishikawa, M.; Nakanishi, T.; Miyazaki, A.; Shimizu, A. Amyloid: Int. J. Exp. Clin. Invest. 1999, 6, 183-186. (11) Costa, P. P.; Figueira, A. S.; Bravo, F. R. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 4499-4503. (12) Skinner, M.; Cohen, A. S. Biochem. Biophys. Res. Commun. 1981, 99, 13261332. (13) Pras, M.; Prelli, F.; Franklin, E. C.; Frangione, B. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 539-542.

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variants has been estimated to be between 1:100 000 and 1:1 000 000 in the United States.2 Certain amino acid substitutions in TTR are hypothesized to affect the stability of the tetramer and cause the TTR to form intermediates that potentially could self-associate into amyloid fibrils.14-18 More than 80 TTR variants have been identified, with the majority being amyloidogenic.19,20 The clinical features of ATTR appear to be related to specific mutations that are characteristic of certain ethnic groups. However, in general, the main clinical characteristics of ATTR are peripheral and autonomic neuropathy, cardiomyopathy, and vitreous opacities. The age of onset depends mainly on the mutation involved. The disease is usually fatal within 7-15 years after the appearance of symptoms.21 Since the most effective treatment of ATTR is liver transplantation,21,22 correct diagnosis is crucial. Mass spectrometry (MS) has played an important role in the clinical diagnosis of ATTR.23,24 Typically, the intact TTR or its tryptic digest is analyzed using electrospray ionization (ESI)25 MS with or without liquid chromatography (LC)26-31 or by matrixassisted laser desorption/ionization (MALDI)32 time-of-flight (TOF) MS.31,33-36 Although LC/MS increases the specificity and improves the signal-to-noise ratio in the analysis, it is time-consuming. LC/ MS requires column equilibration and a blank run to ensure that (14) Lai, Z.; Colo´n, W.; Kelly, J. W. Biochemistry 1996, 35, 6470-6482. (15) Schormann, N.; Murrell, J. R.; Benson, M. D. Amyloid: Int. J. Exp. Clin. Invest. 1998, 5, 175-187. (16) Olofsson, A.; Ippel, H. J.; Baranov, V.; Ho ¨rstedt, P.; Wijmenga, S.; Lundgren E. J. Biol. Chem. 2001, 276, 39592-39599. (17) Quintas, A.; Vaz, D. C.; Cardoso, I.; Saraiva, M. J. M.; Brito, R. M. M. J. Biol. Chem. 2001, 276, 27207-27213. (18) Serag, A. A.; Altenbach, C.; Gingery, M.; Hubbell, W. L.; Yeates, T. O. Biochemistry 2001, 40, 9089-9096. (19) Connors, L. H.; Richardson, A. M.; The´berge, R.; Costello, C. E. Amyloid: Int. J. Exp. Clin. Invest. 2000, 7, 54-69. (20) Saraiva, M. J. M. Hum. Mutat. 2001, 17, 493-503. (21) Bergethon, P. R.; Sabin, T. D.; Lewis, D.; Simms, R. W.; Cohen, A. S.; Skinner, M. Neurology 1996, 47, 944-951. (22) Pomfret, E. A.; Lewis, W. D.; Jenkins, R. L.; Bergethon, P.; Dubrey, S. W.; Reisinger, J.; Falk, R. H.; Skinner, M. Transplantation 1998, 65, 918-925. (23) Wada, Y.; Matsuo, T.; Katakuse, I.; Suzuki, T.; Azuma, T.; Tsujino, S.-I.; Kishimoto, S.; Matsuda, H.; Hayashi, A. Biochim. Biophys. Acta 1986, 873, 316-319. (24) Suzuki, T.; Azuma, T.; Tsujino, S.; Mizuno, R.; Kishimoto, S.; Wada, Y.; Hayashi, A.; Ikeda, S.; Yanagisawa, N. Neurology 1987, 37, 708-711. (25) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (26) Ando, Y.; Ohlsson, P.-I.; Suhr, O.; Nyhlin, N.; Yamashita, T.; Holmgren, G.; Danielsson, A° .; Sandgren, O.; Uchino, M.; Ando, M. Biochem. Biophys. Res. Commun. 1996, 228, 480-483. (27) Kishikawa, M.; Nakanishi, T.; Miyazaki, A.; Shimizu, A.; Nakazato, M.; Kangawa, K.; Matsuo, H. J. Mass Spectrom. 1996, 31, 112-114. (28) Ando, Y.; Ando, E.; Ohlsson, P.-I.; Olofsson, A.; Sandgren, O.; Suhr, O.; Terazaki, H.; Obayashi, K.; Lundgren, E.; Ando, M.; Negi, A. Amyloid: Int. J. Exp. Clin. Invest. 1999, 6, 119-123. (29) Kishikawa, M.; Nakanishi, T.; Miyazaki, A.; Shimizu, A. Amyloid: Int. J. Exp. Clin. Invest. 1999, 6, 48-53. (30) Kishikawa, M.; Nakanishi, T.; Miyazaki, A.; Shimizu, A.; Kusaka, H.; Fukui, M.; Nishiue, T. Amyloid: Int. J. Exp. Clin. Invest. 1999, 6, 278-281. (31) The´berge, R.; Connors, L.; Skinner, M.; Skare, J.; Costello, C. E. Anal. Chem. 1999, 71, 452-459. (32) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (33) Tachibana, N.; Tokuda, T.; Yoshida, K.; Taketomi, T.; Nakazato, M.; Li, Y.F.; Masuda, Y.; Ikeda, S.-I. Amyloid: Int. J. Exp. Clin. Invest. 1999, 6, 282288. (34) The´berge, R.; Connors, L. H.; Skinner, M.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2000, 11, 172-175. (35) Bergquist, J.; Andersen, O.; Westman, A. Clin. Chem. 2000, 46, 12931300. (36) Terazaki, H.; Ando, Y.; Nakamura, M.; Obayashi, K.; Misumi, S.; Shoji, S.; Yamashita, S.; Nakagawa, K.; Ishizaki, T.; Suhr, O.; Uemoto, S.; Inomata, Y.; Tanaka, K. Transplantation 2001, 72, 296-299.

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the column is clean and often takes 60 min per run. In addition, determination of the mass of each peptide in an LC/MS chromatogram requires deconvolution of signals resulting from multiple charge states of the peptide. Furthermore, while analysis of TTR tryptic digest by MALDI-TOF MS is rapid and simple, it does not provide complete sequence coverage, due partly to ion suppression. As a result, a critical peptide containing the modification under investigation may not be detected readily. To circumvent these difficulties, we have developed a multifaceted approach consisting of mass spectrometric peptide mapping with multiple proteases to fully characterize TTR variants in ATTR and direct DNA sequence analysis to confirm these results. Intact TTR is immunoprecipitated from the serum of each patient and purified using high-performance liquid chromatography (HPLC). Nanospray ionization37 MS of the intact TTR shows the mass difference between the wt and variant TTR. To achieve complete sequence coverage and improve the chance of detecting the critical peptide containing the modification, aliquots of immunoprecipitated TTR samples are digested with trypsin, lysyl endopeptidase Lys-C, or endoproteinase Asp-N. MALDI-TOF MS of these TTR enzymatic digests narrows the location of the modification to the span of an individual peptide derived from the variant protein. The precise position and characteristic of the modification in the TTR variant are then established using ESI or MALDI tandem mass spectrometry (MS/MS) and accurate mass measurements. The results are verified by direct DNA sequence analysis of the appropriate TTR exon. Using these methodologies for the analyses of patients’ samples, we have recently identified two nonpathologic variants (Thr119Met and Gly6Ser), three rare but previously reported pathologic variants (Phe64Leu, Asp38Ala, Phe44Ser), and a novel pathologic variant (Trp41Leu). The experimental results that led to these assignments are presented herein. Over the last four years, we have used MS to analyze samples from 182 patients and have found that 65 of these exhibited 28 different variants, including 8 that were previously unreported. EXPERIMENTAL SECTION Materials. All reagents were ACS grade or better. Trifluoroacetic acid (TFA) was purchased from Fluka Chemical Corp. (Milwaukee, WI), formic acid was from EM Science (Gibbstown, NJ), modified trypsin and endoproteinase Asp-N (both sequencing grade) were from Roche Molecular Biochemicals (Indianapolis, IN), lysyl endopeptidase Lys-C was from Wako BioProducts (Richmond, VA), and porcine renin substrate tetradecapeptide was from Sigma Chemical Co. (St. Louis, MO). Ammonium bicarbonate, glacial acetic acid, acetonitrile, and methanol were acquired from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). All solutions were prepared with in-house reverse osmosis purified water that had been further purified with a Hydro Picotech 2 water purification system (Research Triangle Park, NC). Isolation and Purification of TTR from Patient’s Serum. Immunoprecipitation was used to isolate TTR from serum.27 Typically, 160 µL of goat antihuman prealbumin (Diasorin, Stillwater, MN) was mixed with 200 µL of serum from the patient and incubated at 37 °C overnight. The mixture was spun at 14 000 rpm at room temperature for 20 min. The pellet containing the (37) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8.

TTR-antibody complex was collected and washed with purified water three times. Reversed-phase HPLC, with mobile phase A consisting of 0.1% TFA in water and mobile phase B containing acetonitrile with 0.1% TFA, was used to separate the TTR from the antibody. The TTR-antibody complex was dissolved in 96: 2:2 (v/v/v) of water/acetonitrile/acetic acid, applied to an analytical Vydac C-4 column (25 × 0.46 cm, 5-µm particle size, Hesperia, CA), and eluted at 0.75 mL/min over 30 min using a gradient of 40-85% mobile phase B. Elution of the TTR was monitored at 214 nm. TTR eluted at 52-54% mobile phase B. The solvent mixture was removed from the protein using a Savant Instruments SC110 Speedvac concentrator (Holbrook, NY). To avoid the appearance of peaks corresponding to the peptides originating from the antibody used in immunoprecipitation, the conditions reported earlier27,31 have recently been modified. A solvent mixture consisting of 80:10:10 (v/v/v) water/ acetonitrile/acetic acid is now used to dissolve the TTR-antibody complex. Before HPLC purification, the TTR is separated from the antibody using a Microcon YM-100 centrifugal filter (Millipore Corporation, Bedford, MA). Enzymatic Digestions of TTR. Trypsin (enzyme/substrate, 1:100), Asp-N (1:300), and Lys-C (1:100) digestions were carried out in 100 mM ammonium bicarbonate buffer at pH 8 at 37 °C from 5.5 to 18 h. Each enzymatic digestion was quenched by drying the reaction mixture in a Savant Instruments SC110 Speedvac concentrator. Electrospray Ionization Mass Spectrometry. Nanospray needles were pulled from thin-wall borosilicate glass capillaries (1.2-mm o.d., 0.90-mm i.d.; World Precision Instruments, Sarasota, FL) using a Sutter Instrument P 80/PC micropipet puller (San Rafael, CA). The tip of the nanospray needle was approximately 1-2-µm i.d.. ESI mass spectra of the intact TTR proteins were obtained in the positive ion mode using a Micromass Quattro II triple quadrupole mass spectrometer (Beverly, MA) equipped with a nanospray Z-Spray source, scanned over a range of mass-to-charge ratio m/z 150-2000. This mass spectrometer was calibrated using sodium trifluoroacetate ion clusters.38 After calibration, the instrument was capable of achieving at least 0.007% mass accuracy for the deconvoluted mass spectrum. Typically, 2 µL of a 2 µM solution of the immunoprecipitated and HPLC-purified TTR in 50: 50:0.1 (v/v/v) of methanol/water/acetic acid was loaded into a nanospray needle. A stainless steel wire (type 304V, 0.127 mm, 30 gauge; Small Parts Inc., Miami Lakes, FL) was inserted into the nanospray needle containing the sample solution to provide electrospray. The capillary potential was increased slowly from 0 to 1.2 kV until a stable ion current was observed. The cone voltage and ion source temperature were held at 34 V and 82 °C, respectively. The resulting mass spectrum, which contains a distribution of charge states for each protein species in the sample, was obtained by summing a minimum of 10 scans. Each mass spectrum was deconvoluted using the Micromass MassLynx (version 3.4) software to obtain the molecular mass of each protein. To obtain sequence information, the peptide mixture resulting from an enzymatic digest was nanosprayed into the mass

spectrometer and characterized by MS/MS analysis. The [M + 2H]2+ corresponding to the peptide of interest was isolated using the first quadrupole (Q1) and fragmented via collision-induced dissociation (CID) inside the collision cell (Q2), which was held at an Ar pressure of 3.8 × 10-3 Torr. The collision energy was set between 18 and 25 eV to reduce the relative abundance of the precursor ion to about one-third of its original intensity. The product ion spectrum was obtained by scanning Q3. For samples requiring high sensitivity and improved mass accuracy, MS/MS analysis was performed using an Applied Biosystems/MDS-Sciex QSTAR Pulsar quadrupole/orthogonal acceleration TOF mass spectrometer in the positive ion mode. The instrument was calibrated using the [M + 2H]2+ ion (m/z 879.9704) and [M + 4H]4+ ion (m/z 440.4892) of porcine renin substrate tetradecapeptide. For MS/MS experiments, the instrument was calibrated using the b8 (m/z 1028.5317) and y2 (m/z 269.1137) fragment ions of the [M + 3H]3+ ion (m/z 586.9829) of porcine renin substrate tetradecapeptide. After calibration, this mass spectrometer was capable of achieving ∼10 ppm mass accuracy in both MS and MS/MS modes with a minimum resolution of 1:9000 (fwhm). Typically, 3 µL of a 1 µM solution of TTR enzymatic digest in 50:50:1 (v/v/v) of methanol/water/formic acid was loaded into a nanospray needle. A stainless steel wire (type 304V, 0.127 mm, 30 gauge; Small Parts Inc.) was inserted into the nanospray needle containing the sample solution. The capillary potential was increased slowly from 0 to 1.2 kV until a stable ion current was observed. The declustering potential was held at 35 V. The [M + 2H]2+ of the peptide of interest was isolated using the first quadrupole (Q1) and fragmented via CID inside the collision cell (Q2) with an N2 pressure of 3.5 × 10-5 Torr. The collision energy was set between 18 and 28 eV to reduce the relative abundance of the precursor ion to about one-third of its original intensity. The fragment ions were mass analyzed in the TOF region. For accurate mass measurements, the peptide mixture resulting from an enzymatic digest was dissolved in 49:49:2 (v/v/v) of methanol/water/formic acid and analyzed by Fourier transform ion cyclotron resonance (FTICR) MS using a modified IonSpec HiResESI mass spectrometer (Irvine, CA) equipped with a 7-T active-shielded superconducting electromagnet and a home-built nanospray ion source. The sample solution was nanosprayed from New Objective tips (Cambridge, MA) by grounding the solution and ramping the orifice potential to approximately 600-800 V negative relative to the solution. The ion source conditions were adjusted to optimize signal intensity and minimize in-source fragmentation. A standard FTMS pulse sequence was employed to generate broadband mass resolving power in excess of 1:25 000 (fwhm). Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. MALDI-TOF mass spectra of the TTR enzymatic digests were acquired in the positive ion mode using a Finnigan MAT Vision 2000 MALDI-TOF reflectron mass spectrometer (Thermo BioAnalysis Corp., Santa Fe, NM) equipped with a Laser Science nitrogen laser (Franklin, MA) having a 3-ns pulse width at 337 nm. All mass spectra were obtained in the linear mode with delayed extraction39,40 to achieve maximum sensitivity and

(38) Moini, M.; Jones, B. L.; Rogers, R. M.; Jiang, L. J. Am. Soc. Mass Spectrom. 1998, 9, 977-980.

(39) Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, 1998-2003. (40) Vestal, M. L.; Juhasz, P.; Martin, S. A. Rapid Commun. Mass Spectrom. 1995, 9, 1044-1050.

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to avoid the effect of metastable decomposition41,42 of peptides bearing labile modifications. The intensity of the laser shot was set slightly above threshold. A 20-kV accelerating potential was applied to the sample target. For delayed extraction, a 3.4-kV pulse was applied to the probe 450 ns after the laser pulse. Then, the ions were extracted by applying 17 kV to the first extraction lens. The instrument was calibrated with a standard peptide mixture (Agilent Technologies, Palo Alto, CA) consisting of oxytocin [M + H]+ m/z 1008.2, Arg-8-vasopressin [M + H]+ m/z 1085.2, angiotensin I [M + H]+ m/z 1282.5, somatostatin [M + H]+ m/z 1638.9, chicken atrial natriuretic peptide [M + H]+ m/z 3161.7, human recombinant insulin [M + H]+ m/z 5808.7, and recombinant hirudin [M + H]+ m/z 6964.5. After external calibration, this mass spectrometer was capable of achieving ∼0.1% mass accuracy in the linear mode with delayed extraction. Where greater mass accuracy was needed, the instrument was internally calibrated using known ions of wt TTR peptides to obtain at least 0.01% mass accuracy. Typically, 0.5 µL of a 10 pmol/µL solution of a TTR enzymatic digest was mixed on the sample target with 0.5 µL of 15 mg/mL solution of the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) (Agilent Technologies) dissolved in 30:70:0.1 (v/v/ v) of acetonitrile/water/TFA. The mixture was allowed to air-dry. Each MALDI-TOF mass spectrum was obtained by summing the signals acquired from 50 laser shots. Direct DNA Sequence Analysis. Total genomic DNA was isolated from peripheral blood leukocytes. The coding sequence of each exon was amplified using forward and reverse pairs of 21-base oligonucleotide primers as follows: exon 2, 5′-TCTTGTTTCGCTCCAGATTTC-3′, 5′-CAGATGATGTGAGCCTCTCTC3′; exon 3, 5′-CCTACTTCTGACTTAGTTGAG-3′, 5′-ACTGTGCATTTCCTGGAATGC-3′; and exon 4, 5′-GTCATGTGTGTCATCTGTCAC-3′, 5′-CATATGAGGTGAAAACACTGC-3′ (Integrated DNA Technologies, Inc, Coralville, IA). Polymerase chain reaction (PCR) was performed through 39 cycles in this manner: denaturation at 94 °C for 30 s, annealing at 61 °C for 1 min, and extension at 72 °C for 30 s. After electrophoresing on 1.5% agarose gels and staining with ethidium bromide, the PCR products were purified using QIAquick PCR purification kit (Qiagen Inc, Valencia, CA) and sequenced in both directions, using the same oligonucleotides as the primers. Dideoxy terminator florescent DNA sequencing reactions were carried out using the ABI PRISM BigDye Terminators cycle sequencing kit (Applied Biosystems, Foster City, CA). The resulting products were purified by ethanol precipitation and electrophoresed on the ABI PRISM 377 DNA sequencer at the Molecular Genetics Core at Boston University School of Medicine. RESULTS AND DISCUSSION General Strategy for the Analysis of TTR Variants. Our approach for detecting and characterizing TTR variants in our population of patients who may have amyloidosis consists of three phases. The first phase involves an initial isoelectric focusing (IEF) screening to detect the presence of TTR variants in serum.43 Normally, only one band corresponding to the wt TTR is detected (41) Tang, X.; Ens, W.; Standing, K. G.; Westmore, J. B. Anal. Chem. 1988, 60, 1791-1799. (42) Annan, R. S.; Carr, S. A. Anal. Chem. 1996, 68, 3413-3421. (43) Connors, L. H.; Ericsson, T.; Skare, J.; Jones, L. A.; Lewis, W. D.; Skinner, M. Biochim. Biophys. Acta 1998, 1407, 185-192.

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by IEF. The appearance of two bands in the IEF gel suggests the presence of a TTR variant. The TTR is then immunoprecipitated from the serum of the patient, HPLC-purified, and analyzed by MS in the second phase of analysis.31 ESI MS of intact TTR yields the mass difference between the wt and variant TTR proteins. To locate the site of the modification, an aliquot of the TTR sample is treated with a proteolytic enzyme, and the digest mixture is analyzed using MALDI-TOF MS. Finally, in the third phase, the identity and position of the modification in the TTR variant are established unambiguously by MS/MS analysis, accurate mass measurements, and direct DNA sequence analysis. Knowledge of the site of the modification from mass spectrometric analyses simplifies DNA sequence analysis because only the appropriate TTR exon needs to be amplified by PCR and sequenced. Identification of Thr119Met TTR Variant. The masses of the intact TTR proteins were determined using an ESI triple quadrupole mass spectrometer. The deconvoluted mass spectrum of the sample containing the TTR proteins immunoprecipitated and HPLC-purified from the serum of a patient (TTR-99008) is shown in Figure 1. The clinical data of this patient and all other patients discussed herein are shown in Table 1. The deconvoluted mass observed at 13 762 ( 1 Da is in good agreement with the mass calculated (Mcal) from the amino acid sequence of the wt TTR protein at 13 761 Da.44 The deconvoluted masses observed at 13 842 ( 1 and 13 880 ( 1 Da have been assigned to the wt S-sulfated29,31 (Mcal ) 13 841 Da) and S-cysteinylated27 TTR (Mcal ) 13 881 Da), respectively. Furthermore, the deconvoluted masses seen at 13 716 ( 1, 13 938 ( 1, and 14 068 (1 Da are consistent with the Mcal of the wt TTR having its Cys10 side chain cleaved (Mcal ) 13 715 Da), conjugated with cysteinylglycine (Mcal ) 13 938 Da), and conjugated with glutathione (Mcal ) 14 069 Da), respectively.27,29 Each of these wt TTR peaks was accompanied by another peak having an observed mass shift of +30 ( 2 Da, indicating the presence of a modification in the TTR sequence. The peak observed at the deconvoluted mass 13 880 Da is wider than the other peaks in the spectrum and appears to contain unresolved peaks corresponding to both the S-cysteinylated wt TTR and the S-sulfated variant TTR. The +30-Da shift from 13 842 ( 1 Da would yield a peak at 13 872 Da (Mcal ) 13 872 Da), which the triple quadrupole mass spectrometer could not resolve from the peak at 13 880 ( 1 Da containing the S-cysteinylated wt TTR. To locate and identify the modification, an aliquot of the TTR was first digested with trypsin and analyzed using MALDI-TOF MS. The MALDI-TOF mass spectrum shown in Figure 2A matches that of a tryptic digest of wt TTR. The assignment of each [M + H]+ ion to a corresponding peptide resulting from the tryptic digest is summarized in Table 2. These results show that trypsin digestion of TTR typically provides less than full sequence coverage (∼58%). Since no pair of peaks separated by the +30-Da mass shift is present in this spectrum, it was clear that the TTR tryptic peptide containing the modification was not being observed. In a second attempt to locate the modification and obtain more extensive sequence coverage, another aliquot of the TTR sample was digested with Lys-C. The MALDI-TOF mass spectrum of this Lys-C digest is shown in Figure 2B. Table 3 summarizes the (44) Kanda, Y.; Goodman, D. S.; Canfield, R. E.; Morgan, F. J. J. Biol. Chem. 1974, 249, 6796-6805.

Figure 1. Deconvoluted ESI mass spectrum, obtained using the triple quadrupole mass spectrometer, of intact TTR immunoprecipitated and HPLC purified from the serum of patient TTR-99008. The +30-Da shift of the S-sulfated TTR peak from 13 842 ( 1 Da would yield a peak at 13 872 Da, which is not resolved from the S-cysteinylated wt TTR peak at 13 880 ( 1 Da. For other peak assignments, please see text. Table 1. Summary of Patients’ Clinical Data patient

age

sex

TTR-99008a

Thr119Met

50

F

French

- (abdominal fat)

ATTR-99027a

Phe64Leu

73

M

Italian

TTR-00150

Gly6Ser

32

M

English

ATTR-01124

Asp38Ala

69

F

Korean

ATTR-01108

Phe44Ser

37

M

ATTR-00108

Trp41Leu

45

F

Lithuanian/ German Russian

+ (nerve) + (abdominal fat) - (muscle) - (abdominal fat) + (heart) + (abdominal fat) + (nerve) + (abdominal fat) + (vitreous) - (abdominal fat)

a

ethnicity

Congo red results (biopsy site)

TTR variant

clinical features evaluation for diarrhea, father had amyloidosis, type unknown peripheral sensory neuropathy at age 70 evaluation for muscle pain congestive heart failure at age 69 peripheral sensory and autonomic neuropathy at age 36 vitreous opacities at age 43

TTR and ATTR denote that the patient was found to have either a nonpathologic or a pathologic variant, respectively.

assignment of each [M + H]+ ion to a specific peptide resulting from the Lys-C digestion. It can be concluded from these results that Lys-C digestion of this TTR sample produced more peptides that were detected by MALDI-TOF MS. The ions observed at m/z 4950.5 and 5081.2 corresponded to the wt TTR peptides containing residues 81-126 and 81-127, respectively. The +30-Da mass shift of these peptides was detected at m/z 4980.8 and 5111.7, respectively. These results not only confirm the existence of the +30-Da mass shift detected in the ESI mass analysis of the intact TTR proteins but they also suggest that the modification is in the region containing residues 81-126. A mass shift of +30 Da is consistent with any of the following amino acid substitutions: Ala f Thr, Arg f Trp, Gly f Ser, Thr f Met, or Val f Glu. Ions detected at m/z 4997.2 and 5128.1 were m/z 16.4 higher than the ions at m/z 4980.8 and 5111.7, indicating the possible occurrence of oxidation at a Met residue.45,46 No (45) Lagerwerf, F. M.; van de Weert, M.; Heerma, W.; Haverkamp, J. Rapid Commun. Mass Spectrom. 1996, 10, 1905-1910.

Met residue is present in this wt peptide spanning residues 81-126. Thus, the +30-Da mass shift most likely corresponds to introduction of a Met residue via a Thr f Met substitution. A Thr f Met substitution at position 119 would correspond to a single nucleotide transition in the TTR gene, ACG f ATG. Direct DNA sequence analysis of exon 4 of the TTR DNA sample from patient TTR-99008 showed a cytosine to thymine base transition (ACG f ATG) in the codon at position 119 (Figure 3 A), thus confirming the Thr f Met substitution. This nonpathologic variant47 has been shown to stabilize the tetrameric TTR.48,49 (46) Mo, W.; Ma, Y.; Takao, T.; Neubert, T. A. Rapid Commun. Mass Spectrom. 2000, 14, 2080-2081. (47) Harrison, H. H.; Gordon, E. D.; Nichols, W. C.; Benson, M. D. Am. J. Med. Genet. 1991, 39, 442-452. (48) Alves, I. L.; Hays, M. T.; Saraiva, M. J. M. Eur. J. Biochem. 1997, 249, 662-668. (49) Hammarstro ¨m, P.; Schneider, F.; Kelly, J. W. Science 2001, 293, 24592462.

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Table 2. Peptides Resulting from Trypsin Digestion of an Aliquot of the TTR Sample from Patient TTR-99008 Observed by MALDI-TOF MS (Figure 2A) [M + H]+, m/z residues

obsd

calcd

peptide sequence

1-9 16-21 22-34 35-48 36-48 49-70 71-80 71-76 77-80

834.0 673.0 1367.7 1523.6 1395.5 2455.6 1268.5 704.8 583.7

833.9 672.8 1367.6 1523.6 1395.5 2456.6 1269.4 704.8 583.7

GPTGTGESK VLDAVR GSPAINVAVHVFR KAADDTWEPFASGK AADDTWEPFASGK TSESGELHGLTTEEEFVEGIYK VEIDTKSYWK VEIDTK SYWK

Lys-C digestion of TTR provides ∼95% sequence coverage, and peptides containing 121/127 amino acid residues were observed by MS. In a further attempt to detect the peptide ion(s) containing the remaining six amino acid residues, another aliquot of the TTR sample from the same patient (TTR-99008) was digested with AspN. The MALDI-TOF mass spectrum of this digest is shown in Figure 2C. Table 4 summarizes the assignment of each [M + H]+ ion to a specific peptide resulting from the Asp-N digest. As shown in Table 4, Asp-N digestion of the TTR provided 100% sequence coverage. The ion detected at m/z 3159.5 corresponded to the TTR wt peptide containing residues 99-127 (Figure 2C). A +30Da mass shift of this peptide yielded the peak at m/z 3189.8, revealing the region of the TTR that contains the modification. The ion at m/z 1718.2 corresponded to the TTR wt peptide containing residues 1-17. The S-sulfation and S-cysteinylation of the TTR Cys10 residue were observed at m/z 1797.4 and 1837.1, respectively. The Asp-N digest produced two peptides, each containing the Cys10 residue, that were not seen in either the tryptic or Lys-C digest. Identification of Phe64Leu TTR Variant. ESI mass analysis of the intact TTR proteins immunoprecipitated and HPLC-purified from the serum of a second patient (ATTR-99027) indicated the presence of a variant that is 34 ( 2 Da lower in mass than the wt protein. An aliquot of the TTR sample was digested with Lys-C and analyzed using MALDI-TOF MS. MALDI-TOF mass analysis of this digest showed numerous peptides (Figure 4A). The ions detected at m/z 2456.3 and 3142.7 corresponded to wt TTR

Figure 2. Linear MALDI-TOF mass spectra of the (A) tryptic, (B) Lys-C, and (C) Asp-N digests of unfiltered aliquots of the TTR sample from patient TTR-99008. In panel B, the ions at m/z 4997.2 and 5128.1, marked with an asterisk, are likely due to a Met oxidation. In panel C, the ions at m/z 1797.4 and 1837.1 corresponded to the S-sulfation and S-cysteinylation modifications, respectively, in the peptide containing residues 1-17. Numbers in parentheses correspond to the position of the amino acid residues within the TTR sequence. Unlabeled peaks correspond to peptides originating from the antibody used in immunoprecipitation.

peptides containing residues 49-70 and 49-76, respectively. The -34-Da mass shift of these peptides was detected at m/z 2422.3 and 3108.4, suggesting that the region spanning residues 49-70 contained the modification. A mass shift of -34 Da is consistent with a Met f Pro, His f Cys, Tyr f Glu, or Phe f Leu/Ile amino acid substitution. The substitution could not be Met f Pro

Table 3. Peptides Resulting from Lys-C Digestion of an Aliquot of the TTR Sample from Patient TTR-99008 Observed by MALDI-TOF MS (Figure 2B) [M + H]+, m/z

a

residues

obsd

calcd

peptide sequence

1-9 16-35 16-48 36-48 49-70 49-76 77-80 81-126 81-126a 81-127 81-127a

833.4 2149.0 3527.2 1395.3 2455.7 3142.4 583.0 4950.5 4980.8 5081.2 5111.7

833.9 2149.6 3526.0 1395.5 2456.6 3142.4 583.7 4951.6 4981.6 5080.7 5110.8

GPTGTGESK VLDAVRGSPAINVAVHVFRK VLDAVRGSPAINVAVHVFRKAADDTWEPFASGK AADDTWEPFASGK TSESGELHGLTTEEEFVEGIYK TSESGELHGLTTEEEFVEGIYKVEIDTK SYWK ALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPK ALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTMAVVTNPK ALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE ALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTMAVVTNPKE

Denotes a variant peptide containing the Thr f Met substitution at position 119.

746 Analytical Chemistry, Vol. 74, No. 4, February 15, 2002

Table 4. Peptides Resulting from Asp-N Digestion of an Aliquot of the TTR Sample from Patient TTR-99008 Observed by MALDI-TOF MS (Figure 2C) [M + H]+, m/z

a

residues

obsd

calcd

peptide sequence

1-17 1-17 1-17 18-37 38-73 39-73 74-98 99-127 99-127a

1718.2 1797.4 1837.1 2079.2 4032.3 3918.5 2849.2 3159.5 3189.8

1718.1 1798.2 1837.2 2079.4 4032.3 3917.2 2849.2 3159.5 3189.6

GPTGTGESKCPLMVKVL GPTGTGESKCPLMVKVL,Cys10 is S-sulfated GPTGTGESKCPLMVKVL,Cys10 is S-cysteinylated DAVRGSPAINVAVHVFRKAA DDTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEI DTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEI DTKSYWKALGISPFHEHAEVVFTAN DSGPRRYTIAALLSPYSYSTTAVVTNPKE DSGPRRYTIAALLSPYSYSTMAVVTNPKE

Denotes a variant peptide containing the Thr f Met substitution at position 119.

Figure 3. Direct DNA sequence analyses of (A) exon 4 (patient TTR-99008), (B) exon 3 (patient ATTR-99027), (C) exon 2 (patient TTR-00150), (D) exon 2 (patient ATTR-01124), (E) exon 2 (patient ATTR-01108), and (F) exon 2 (patient ATTR-00108). Peaks are labeled to indicate the nucleotide bases C (O), T (4), A (0), and G (*).

because there are no Met residues in the wt peptide containing residues 49-70. Although there are His and Tyr residues in the wt peptide, the His f Cys or Tyr f Glu substitution is unlikely since these substitutions would require more than a single base substitution in the nucleotide sequence. However, the codon for Phe at position 64 is TTT in the TTR gene sequence. Three of the codons for Leu are TTA, TTG, and CTT, and one of the codons for Ile is ATT. Thus, only a single base substitution in the TTR gene sequence is required for the amino acid to change from Phe to Leu/Ile at positon 64. Direct DNA sequence analysis of exon 3 of the TTR DNA sample from this patient (ATTR-99027) showed a thymine to cytosine base transition (TTT f CTT) in the codon at position 64 (Figure 3B), thus defining the substitution as Phe f Leu. This pathologic variant has been identified previously.50 Identification of Gly6Ser TTR Variant. ESI mass analysis of the intact TTR proteins immunoprecipitated and HPLC purified (50) Ii, S.; Minnerath, S.; Ii, K.; Dyck, P. J.; Sommer, S. S. Neurology 1991, 41, 893-898.

from the serum of a third patient (TTR-00150) indicated the presence of a variant that is 30 ( 2 Da higher in mass than the wt protein. Digestion of an aliquot of the TTR sample with Lys-C and analysis of the resulting peptide mixture using MALDI-TOF MS provided enough sequence coverage to include the region in the TTR that contains the modification (Figure 4B). The ion observed at m/z 833.5 corresponded to the wt peptide containing residues 1-9. The +30-Da mass shift of this peptide was observed at m/z 863.2, suggesting that the region containing residues 1-9 has the modification. A mass shift of +30 Da is consistent with an Ala f Thr, Arg f Trp, Val f Glu, Thr f Met, or Gly f Ser substitution. Since there are no Ala, Arg, and Val residues in the first nine residues of the wt TTR amino acid sequence, the first three of these potential amino acid substitutions can be eliminated. However, it is possible to have a Gly f Ser substitution at position 1 (GGC f AGC) or position 6 (GGT f AGT) or a Thr f Met substitution at position 3 (ACG f ATG), based on a single base substitution in the TTR gene sequence. To identify the amino acid substitution and its position in the sequence, the TTR Lys-C digest mixture was analyzed using an ESI quadrupole/orthogonal acceleration TOF mass spectrometer. The [M + 2H]2+ of the peptides of interest were isolated and fragmented using CID to obtain sequence information. The product ion mass spectrum of the [M + 2H]2+ ion at m/z 417.206 of the wt TTR peptide containing residues 1-9 is shown in Figure 5A. Figure 5B illustrates the product ion mass spectrum of the [M + 2H]2+ ion of the variant peptide at m/z 432.210. The mass difference between sequential fragment ions in the product ion mass spectrum corresponds to the mass of an amino acid residue. The complete sequences of these two peptides could be deduced from the b- and y-fragment ion series.51,52 In both panels A and B of Figure 5, the y1, y2, y3, and y3 - H2O (due to a water loss) fragment ions have the same m/z for both the wt and variant peptides and therefore contain the same amino acid sequences (Glu-Ser-Lys, residues 7-9). The y4 fragment ion of the wt peptide was detected at m/z 420.211, confirming that residue 6 is Gly (y4 - y3 ) 57.049 Da, the mass of a Gly residue). However, the y4 fragment ion of the variant peptide was observed at m/z 450.205, signifying that residue 6 in the variant peptide is Ser (y4 - y3 ) 87.013 Da, the mass of a Ser residue). These MS/MS results (51) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (52) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-111.

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Figure 4. Linear MALDI-TOF mass spectra of the Lys-C digests of unfiltered aliquots of the TTR samples from (A) patient ATTR-99027 and (B) patient TTR-00150.

Figure 5. ESI product ion mass spectra, obtained using the quadrupole/orthogonal acceleration TOF mass spectrometer, of the [M + 2H]2+ ions of the (A) wt peptide at m/z 417.206 and (B) variant peptide at m/z 432.210 from the Lys-C digest of an aliquot of the TTR sample from patient TTR-00150.

reveal the identity and location of the amino acid substitution in the TTR variant, a Gly f Ser substitution at position 6. Direct DNA analysis of TTR exon 2 of the DNA sample from patient TTR-00150 showed a guanine-to-adenine base transition (GGT f 748 Analytical Chemistry, Vol. 74, No. 4, February 15, 2002

AGT) in the codon at position 6 (Figure 3C), consistent with the Gly f Ser substitution. This Gly6Ser TTR variant is nonpathologic.53 Approximately 12% of Caucasians appear to be heterozygous for this variant.54

Figure 6. Linear MALDI-TOF mass spectra of the Lys-C digests of aliquots of the TTR samples from (A) patient ATTR-01124, (B) patient ATTR-01108, and (C) patient ATTR-00108, showing the range m/z 1300-2500.

Identification of Asp38Ala and Phe44Ser TTR Variants. ESI MS of intact TTR immunoprecipitated and HPLC purified from the sera of patients ATTR-01124 and ATTR-01108 showed the presence of a TTR variant that is 44 ( 2 and 60 ( 2 Da lower in mass than wt TTR, respectively. Treatment of aliquots of the TTR samples with Lys-C and analysis of the resulting Lys-C peptide mixtures using MALDI-TOF MS localized the regions containing the modifications that gave rise to the -44- and -60-Da mass shifts (Figure 6A and B). A mass shift of -44 Da is consistent with the following amino acid substitutions: Thr f Gly, Asp f Ala, Met f Ser, and Phe f Cys. In contrast, a mass shift of -60 Da may arise from a Met f Ala, Phe f Ser, or Tyr f Cys substitution. Aliquots of the Lys-C digests were further analyzed by MS/MS using an ESI quadrupole/orthogonal acceleration TOF mass spectrometer to identify the modifications and their positions. Figure 7 shows the product ion mass spectra of the [M + 2H]2+ ions of the wt peptide (from patient ATTR-01108) and the two variant peptides all containing residues 36-48 at m/z 697.820, 675.784 (patient ATTR-01124), and 667.757 (patient ATTR-01108), respectively. In Figure 7B, the residue at position 38 of the variant peptide is undoubtedly Ala: the b2 and b3 fragment ions were detected at m/z 143.080 and 214.107, respectively (b3 - b2 ) 71.027 Da, the mass of an Ala residue), and the y10 and y11 fragment ions were (53) Fitch, N. J. S.; Akbari, M. T.; Ramsden, D. B. J. Endocrinol. 1991, 129, 309-313. (54) Jacobson, D. R.; Alves, I. L.; Saraiva, M. J.; Thibodeau, S. N.; Buxbaum, J. N. Hum. Genet. 1995, 95, 308-312.

observed at m/z 1137.471 and 1208.476 (y11 - y10 ) 71.005 Da, the mass of an Ala residue). In contrast, the residue at position 38 of the wt peptide is Asp (Figure 7A): the b2 and b3 fragment ions were seen at m/z 143.075 and 258.096, respectively (b3 - b2 ) 115.021 Da, the mass of an Asp residue), and the y10 and y11 fragment ions were observed at m/z 1137.462 and 1252.501 (y11 - y10 ) 115.039 Da, the mass of an Asp residue). Direct DNA sequence analysis of exon 2 of the TTR gene from the DNA sample of patient ATTR-01124 showed an adenine-to-cytosine base transversion (GAT f GCT) in the codon at position 38 (Figure 3D), consistent with the Asp f Ala substitution. This Asp38Ala TTR variant is pathologic and has been previously reported.30,33 Similarly, the product ion mass spectrum of the [M + 2H]2+ ion of the variant peptide (patient ATTR-01108) containing residues 36-48 at m/z 667.757 is shown in Figure 7C. The residue at position 44 is clearly Ser (Figure 7C): the y4 and y5 fragment ions were observed at m/z 362.182 and 449.238 (y5 - y4 ) 87.056 Da, the mass of a Ser residue). In contrast, the residue at position 44 of the wt peptide is Phe (Figure 7A): the y4 and y5 fragment ions were seen at m/z 362.195 and 509.250, respectively, (y5 - y4 ) 147.055 Da, the mass of a Phe residue). Direct DNA sequence analysis of TTR exon 2 of the DNA sample from this patient (ATTR-01108) showed a thymine-to-cytosine base transition (TTT f TCT) in the codon at position 44 (Figure 3E), consistent with the Phe f Ser substitution. This pathologic variant has been previously described.55 Identification of a Novel Trp41Leu TTR Variant. ESI mass analysis of the intact TTR proteins immunoprecipitated and HPLC purified from the serum of another patient (ATTR-00108) indicated the presence of a variant that is 73 ( 2 Da lower in mass than the wt protein. An aliquot of the TTR sample was digested with LysC. MALDI-TOF mass analysis of this digest showed many peptides and revealed the region in the TTR that contains the modification (Figure 6C). The ion observed at m/z 1395.7 corresponded to the wt TTR peptide containing residues 36-48. The -73-Da mass shift of this peptide was detected at m/z 1322.3, suggesting that the modification was in the region containing residues 36-48. A mass shift of -73 Da is consistent with a Trp f Leu/Ile substitution. Since the wt peptide containing residues 36-48 contains one Trp residue at position 41, the -73-Da mass shift most likely corresponds to a Trp f Leu/Ile. To verify this observation, the TTR Lys-C digest mixture was analyzed by MS/MS using an ESI triple quadrupole mass spectrometer. The product ion mass spectrum of the [M + 2H]2+ ion of the wt peptide containing residues 36-48 at m/z 698.0 is shown in Figure 8A. Figure 8B illustrates the product ion mass spectrum of the [M + 2H]2+ ion of the variant peptide at m/z 661.4. The complete sequence of these two peptides could be deduced from the detection of the y1-y11 and b1-b7 fragment ion series. These MS/MS results revealed the identity and location of the amino acid substitution, a Trp f Leu/Ile substitution at position 41. The y7 fragment ion of both the wt and variant peptides was detected at m/z 735.4. The y8 fragment ion of the wt peptide was observed at m/z 921.5, indicating that residue 41 is indeed Trp (y8 - y7 ) 186.1 Da, the mass of a Trp residue). However, the y8 fragment ion of the variant peptide was observed at m/z (55) Klein, C. J.; Nakumura, M.; Jacobson, D. R.; Lacy, M. Q.; Benson, M. D.; Petersen, R. C. Neurology 1998, 51, 1462-1464.

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Figure 7. ESI product ion mass spectra, obtained using the quadrupole/orthogonal acceleration TOF mass spectrometer, of the [M + 2H]2+ ions of the (A) wt peptide at m/z 697.820 (from the ATTR-01108 sample), (B) variant peptide at m/z 675.784 (patient ATTR-01124), and (C) variant peptide at m/z 667.757 (patient ATTR-01108) from the Lys-C digests of aliquots of the TTR samples.

848.6, signifying that residue 41 in the variant peptide is either Leu or Ile (y8 - y7 ) 113.2 Da, the mass of a Leu or Ile residue). Detection of the b5 and b6 fragment ions in the two product ion mass spectra confirmed these results. Low-energy CID MS/MS analysis cannot differentiate between the isomeric Leu and Ile residues. Inspection of the TTR exon 2 gene sequence showed that the codon for Trp at position 41 is TGG. One of the codons for Leu is TTG, which would require only a single base substitution in the TTR gene sequence for the amino acid to change from Trp to Leu. On the other hand, the codons for Ile are ATT, ATC, and ATA, which would require a triple base substitution. It thus seemed more likely that the mass shift of -73 Da corresponded to a Trp f Leu substitution at position 41. For accurate mass measurements, another aliquot of the TTR sample was digested with trypsin and analyzed using FTICR mass spectrometry. The relevant mass range shows three isotopic distributions at m/z 725.3667, 747.9335, and 761.8644 (Figure 9). Because they each have an isotope spacing of m/z 0.5, one may conclude that they are [M + 2H]2+ ions. The ions observed at m/z 761.8644 and 747.9335 corresponded to the [M + 2H]2+ ions of peptides containing residues 35-48 (calculated [M + 2H]2+ m/z 761.8628) and 22-35 (calculated [M + 2H]2+ m/z 747.9312), respectively. The ion observed at m/z 725.3667 represented a shift of -36.4977 from the ion at m/z 761.8644. Since these ions are doubly charged (z ) 2), the shift of -36.4977 on the m/z scale must be multiplied by two to obtain the mass shift of -72.9954 Da, which is in excellent agreement with the mass shift calculated for the Trp f Leu/Ile substitution, -72.9952 Da. 750 Analytical Chemistry, Vol. 74, No. 4, February 15, 2002

Direct DNA sequence analysis of exon 2 of the TTR gene from the DNA sample of patient ATTR-00108 showed a guanine-tothymine base transversion (TGG f TTG) in the codon at position 41 (Figure 3F), consistent with the Trp f Leu substitution. This novel Trp41Leu TTR variant was identified simultaneously by the mass spectrometric and direct DNA sequence experiments reported herein and by direct DNA sequencing and RFLP performed at the Indiana University School of Medicine.56 CONCLUSIONS Mass spectrometric peptide mapping of TTR variants is an important tool in the clinical diagnosis of ATTR. It provides a wealth of information in a short period of time and consumes only low-picomole quantities of sample. It reveals the location of the modification in the variant protein. The mass spectral information improves efficiency in DNA sequence analysis because only the exon of the TTR containing the site of the modification needs to be amplified by PCR. The ability to sequence the TTR peptides by MS also facilitates the location and identification of the extensive posttranslational modifications of TTR. Abbreviations: ATTR, familial transthyretin amyloidosis; CID, collision-induced dissociation; DHB, 2,5-dihydroxybenzoic acid; ESI, electrospray ionization; FT, Fourier transform; HPLC, highperformance liquid chromatography; ICR, ion cyclotron resonance; IEF, isoelectric focusing; Mcal, calculated mass; MALDI, matrixassisted laser desorption/ionization; [M + H]+, protonated (56) Yazaki, M.; Connors, L. H.; Eagle, R. C.; Leff, S. R.; Skinner, M.; Benson, M. D., unpublished work.

Figure 8. ESI product ion mass spectra, obtained using the triple quadrupole mass spectrometer, of the [M + 2H]2+ ions of the (A) wt peptide at m/z 698.0 and (B) variant peptide at m/z 661.4 from the Lys-C digest of an aliquot of the TTR sample from patient ATTR-00108.

Figure 9. High-resolution ESI FTICR mass spectrum of the tryptic digest of an aliquot of the TTR sample from patient ATTR-00108, showing the range m/z 720-770. For the discussion of the peak assignments, please see text.

molecule; MS, mass spectrometry; MS/MS, tandem mass spectrometry; m/z, mass-to-charge ratio; PCR, polymerase chain reaction; SCA, senile cardiac amyloidosis; SSA, senile systemic amyloidosis; TFA, trifluoroacetic acid; TOF, time of flight; TTR, transthyretin; wt, wild type. ACKNOWLEDGMENT This work was supported by NIH Grants P41-RR10888 (C.E.C.), S10-RR10493 (C.E.C.), the Gerry Foundation (M.S.), and the Young Family Amyloid Research Fund (M.S.). We thank Alyssa M. Amico, Maurya G. Kaut, and Maxence Metayer-Adams for their assistance in immunoprecipitation. We acknowledge Dr.

Giampaolo Merlini (University of Pavia, Italy) for providing the sequences of the primers for exons 2-4 of the TTR gene and Dr. Terrell T. Gibbs (Department of Pharmacology and Experimental Therapeutics, BUSM) for allowing us access to the micropipet puller for preparation of nanospray tips. We are grateful to Thermo BioAnalysis Corp. and Applied Biosystems for loan of the Vision 2000 and the QSTAR mass spectrometers, respectively.

Received for review July 10, 2001. Accepted November 26, 2001. AC010780+ Analytical Chemistry, Vol. 74, No. 4, February 15, 2002

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