This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Transthyretin (TTR) is a homotetramer with each subunit consisting of 127 amino acid residues. Amino acid substitutions in monomeric TTR are hypothesized to destabilize the tetramer and cause the TTR to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with the deposition of TTR variants as amyloid fibrils in various organs and tissues. Since the most effective treatment of ATTR is liver transplantation, correct diagnosis is crucial. MALDI peptide mapping, ESI tandem MS, and/or accurate mass measurements have been used to identify TTR variants. Top down analysis of Tranthyretin (TTR) wild type, Val30Met and Val122Ile variants and TTR from deposited fibrils was performed by nanospray ESI FTMS using Q2 CAD and SORI-CAD on our hybrid qQq FTMS instrument and on the QStar qoTOF MS instrument (1, 4). TTR patient samples bearing Val30Met and Val122Ile variants were analyzed in this manner. This constitutes a useful clinical application of top down mass spectrometric analysis. TTR samples were obtained by immunoprecipitation (2) from patient sera and from fibrillar deposits. Samples were dissolved in standard ESI buffer (50:50 acetonitrile/water with 0.1% formic acid). The preliminary results obtained by applying top down analysis to TTR variant characterization yield extensive sequence information. The 15+ charge state of the protein was pre-selected for CAD in the second quadrupole prior to mass analysis of the fragments in the ICR cell. The main fragments observed were b42 and y85. These b and y fragments are complementary pairs covering the whole protein sequence. The dominance of the fragment ion spectrum by these fragments is easily understood as b42 and y85 result from a glutamic acid-proline cleavage between positions 42 and 43. Although we are exploring conditions to improve sequence coverage of TTR using Q2 CAD, we have also pursued an approach where the main fragments generated by Q2 CAD, b42 and y85, were isolated in the FTMS cell and made to undergo SORI-CAD to yield sequence information. This two stage approach was also used for a TTR sample containing a Val30Met variant. The variant and wild type protein were pre-selected for Q2 CAD. The Q2 CAD spectrum of the protein exhibited the b42 fragment and a +32 Da peak not present in the wild type TTR Q2 CAD spectrum and consistent with the Val30Met mutation. The isolation and SORI CAD fragmentation of the b42 fragment bearing the variant allowed a more precise location of the variant to positions 19-32 but did not yield data that specified the mutation site. A similar approach was successfully used to characterize a Val122Ile mutation by isolating y85 produced from Q2 CAD of m/z 924 to undergo SORI CAD to yield sequence information locating the mutation. An immunoglobulin light chain involved in primary amyloidosis (AL) was also investigated. The protein isolated from the urine of a patient was analyzed using the same method that was applied to TTR variants. Q2 CAD followed by SORI CAD was necessary to generate fragmentation of this 23 kDa protein, probably due to the presence of intermolecular disulfide bonds (Cys23 to Cys88 and Cys134 to Cys194). Fragmentation was observed to occur almost exclusively the middle of the molecule. Recent experiments on the Sciex Q-Star Q-o-TOF MS have demonstrated that very similar fragmentation patterns can be obtained on that instrument (4). The availability of the FTMS data facilitates interpretation of the Q-o-TOF MS spectra. Ongoing experiments are evaluating results from the recently installed Thermo-Fisher LTQ-Orbitrap MS for this type of sequencing.1. J. L. Pittman, B. A. Thomson, B.A. Budnik, J.J. Cournoyer, E. Fallows, J.A. Jebanathirajah, S.C. Moyer, C.E. Costello, P.B. O'Connor, Proceedings of the 52nd Conference of the ASMS 2004.2. A. Lim, T. Prokaeva, M. McComb, P. B. O'Connor, R. Th berge, L. H. Connors, M. Skinner, and C. E. Costello, Anal. Chem. 74 (2002) 741-751.3. S.H. Guan, A.G. Marshall Int. J. Mass Spectrom Ion Process 158 (1996) 5-37.4. J. S. Kingsbury, R. Theberge, J. A. Karbassi, A. Lim, C. E. Costello, L. H. Connors. .Anal Chem. 79 (2007) 1990-1998.
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