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. 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 posttranslationally 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 (e.g., 00-131 and 00-051, discussed herein) isolated from the urine of patients diagnosed with AL amyloidosis, 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 an important role in destabilizing the folding state of these proteins, thus making them amyloidogenic. AFM is used to observe fibrils. The molecular masses of the intact protein are determined by 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. These tandem MS experiments are also employed to acquire further information on PTMs. Results and Discussion The urinary immunoglobulin LC from a patient (00-131) was purified using dialysis and gel filtration chromatography. Its molecular weight did not include any components that matched the MW deduced from the cDNA sequence. After reduction, two components of molecular weights 23202 and 23248 were detected. The component of molecular weight 23248 matches that deduced from the cDNA sequence whilst 23202 is 80 Da less than the main component in the non reduced ESI-MS spectrum. HPLC MSMS analysis of the Glu-C digest of the protein indicates that the C-terminal cysteine is primarily S-sulfonated, explaining the -80 Da shift observed after reduction. This is the only the second example of S-sulfonated human protein. We have previously reported the first case in transthyretin (2). Before reduction, the deconvoluted ESI mass spectrum of LC 00-051 showed one major peak at 24,182 Da. After treatment with DTT, another major peak at 24,154 Da was observed, which was in good agreement with the theoretical mass of 24,156 Da calculated from the cDNA-deduced amino acid sequence. In addition, two low-abundance, lower-mass components were present in the reduced sample. The cDNA-deduced sequence was verified by MS peptide mapping and MS/MS sequencing with full sequence coverage obtained. Two disulfide bonds (Cys23-Cys94, Cys140-Cys200) were identified and low abundance S-sulfonation, as well as novel modifications to both the N- and C-termini were elucidated. Our AFM results show that for sample 00-131, the protein aggregates with time. There are also clear pH effects during the protein aggregation process although no fibril formation was observed under the conditions used. These results are being compared with those obtained for other sequenced amyloid-forming immunoglobulin LCs exhibiting posttranslational modifications such as homodimerization, S-cysteinylation, N-terminal modifications and glycosylation. 1. Lim A, Wally J, Walsh MT, Skinner M, Costello CE. Anal. Biochem., 2001, 295, 45-56. 2. Th¿berge R, Skinner M, Connors LH, Skare J., Costello CE, Anal.Chem, 1999, 71 452-459.