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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Post-translational modifications at cysteine may be involved in making certain proteins amyloidogenic. We are investigating the correlation of such PTMs on two proteins that have great interest for the amyloid diseased 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 substitution in TTR is 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. 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 and LTQ-Orbitrap MS/MS with emphasis on on-line information dependent acquisition (IDA) capillary LC MS/MS for the characterization of TTR via automated data-base 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 MALDI- and capLC-ESI-QoTOFMS/MS are performed on an Sciex/AB QSTAR QoTOF mass spectrometer. On-line capillary LC-MS with information dependent acquisition (IDA) MS/MS is performed on the QStar and LTQ-Orbitrap systema. 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. 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.