PROTEIN CYSTEINE POST-TRANSLATIONAL MODIFICATION IN AMYLOIDOSIS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Post-translational modifications at cysteine may be involved in making certain proteins amyloidogenic. We are investigating the correlation of such PTMs on two types of proteins that have great interest for the amyloid diseases that are especially central to the patient population seen at the BUSM Amyloid Treatment and Research Center, the serum protein transthyretin and the overexpressed immunoglobulin light chains. Transthyretin (TTR) is a 13.7-kDa transport protein which is synthesized predominantly by the liver. In plasma, tetrameric TTR binds retinol-binding protein and thyroxine. Amino acid substitutions in TTR, and/or post-translational modifications are hypothesized to destabilize the tetramer and cause the TTR to form an intermediate that self associates into amyloid fibrils. Familial transthyretin amyloidosis (ATTR), is associated with the deposition of the TTR variants as amyloid fibrils in various tissues and organs. More than 90 TTR variants have been identified, with the majority being amyloidogenic. Senile systemic amyloidosis is associated with deposition of the wt protein and becomes common (>25%) in patinets over 80 yr. Since the only effective treatment of ATTR is liver transplantation, the correct clinical diagnosis is critical. The MS Resource, in collaboration with the Amyloid Treatment and Research Program, has characterized a number of TTR variants of clinical significance. We are now exploring the use of QoTOF MS/MS., LTQ-Orbitrap MS/MS and FTMS/MS with emphasis on on-line information dependent acquisition (IDA) nano LC MS/MS for the characterization of TTR via automated database searching. TTR is immunoprecipitated from the serum of patients and purified by centrifugation and reversed phase HPLC. Proteolytic digestions are performed and the digests are first analyzed by MALDI-TOFMS. On-line capillary and nanoLC-MS with information dependent acquisition (IDA) MS/MS is performed on the QStar and LTQ-Orbitrap systems. Data from IDA-LC-MS/MS are analyzed against Mascot (Matrix Science) and user programmed PRO-ID (ABI) databases. Mass spectrometry (MS) has played an important role in the clinical diagnosis of ATTR. Since manual collection and interpretation of ESI/MALDI/MS/MS data is time consuming and inefficient, use of proteomics developed MS/MS methods, such as IDA-LC-MS/MS with automated database searching, offers advantages to the clinical applications of mass spectrometry. New separation methods are being evaluated to see whether they may offer advantages. Conclusive variant identification is obtained in cases where the mutation has been pre-programmed into the database.and automated assignment of PTMs such as modifications at cysteine can be handled using the preprogrammed database. The same approach is being developed for cysteine-modified IgG light chains. Chemical tools are also being developed to aid in determination of unusual modifications that may accompany cysteinylation.