MS CHAR OF AMYLOIDOGENIC LIGHT CHAINS OF PTS DIAGNOSED W/PRIMARY AMYLOIDOSIS

点击翻译按钮获取中文摘要

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. Systemic Primary (AL, immunoglobulin light chain) amyloidosis is characterized by the deposition of immunoglobulin light chain (LC) proteins produced by a monoclonal B-cell-derived clone. Most of the previously analyzed LCs have been found to be post-translationally modified (1). The most common case is S-cysteinylation of the C-terminal cysteine in addition to the intramolecular disulfide bonds normally found in immunoglobulin LCs. Our previous studies have shown that the amino acid replacements in the variable region and some post-translational modifications (PTMs) of immunoglobulin LCs may be the key factors that contribute to fibril formation by destabilizing the folding state of these proteins. To have a better understanding of the role of LC modifications on amyloid deposition, we use a mass spectrometry (MS) based method to investigate amyloidogenic LCs isolated from the urine of patients diagnosed with AL amyloidosis, as well as 2D-gel separation followed by in-gel digestion and nanoESI LC-MS/MS, to identify amino acid sequence variations and PTMs in the protein. The folding stability of an immunoglobulin LC is generally regarded as a controlling key of its tendency to form amyloid fibrils; some amino acid replacements and PTMs of LCs may play important roles in destabilizing the folding state of these proteins, thus making them amyloidogenic. AFM is used to observe fibrils obtained from patient tissues and others grown in vitro under various conditions. The molecular masses of the intact protein are determined by MALDI-MS and/or nanospray ESI-MS, before and after treatment with dithiothreitol (DTT). The sample aliquots are proteolytically digested with trypsin, Asp-N, Lys-C, and Glu-C. Aliquots of these enzymatic digestion products are reduced with DTT. MALDI-MS and ESI-MS analyses of both reduced and non-reduced digests are performed to generate peptide maps. Some peptides that cannot be assigned by peptide mapping are sequenced using ESI-MS/MS or MALDI MS/MS on a quadrupole orthogonal TOF MS and/or the LTQ-Orbitrap MS system. These tandem MS experiments are also employed to acquire further information on PTMs. Our AFM results show that the LC protein aggregates with time. There are also clear pH effects during the protein aggregation process. These results from different patient samples are being compared; the samples contain many post-translational modifications such as homodimerization, S-cysteinylation, N-terminal modifications and glycosylation. The specificity of interactions with glycosaminoglycans is being investigated as well.