Abstractβ-2-microglobulin (β2m) self-associates into fibrillar amyloid deposits in the musculoskeletal system of patients undergoing hemodialysis treatment. Previous studies have shown that stoichiometric amounts of Cu(II) at near physiological conditions can cause β2m to organize into native-like dimers prior to forming amyloid fibrils. Here, we report the results from selective covalent labeling reactions combined with mass spectrometry that provide insight into the amino acid residues that mediate dimer formation in the wild-type protein. Using three complementary covalent labeling reagents, we find that the dimer interface is formed by the anti-parallel stacking of ABED β-sheets from two β2m monomers. In addition, our data clearly indicate that a dimer interface involving the interactions of D-D strands from separate protein units as seen in the recent crystal structures of two mutant β2m oligomers is unlikely.β-2-microglobulin (β2m) is the non-covalently bound light chain of the class I major histocompatibility complex (MHC-I) (1). It is a monomeric protein with 99 residues (~12 kDa). It adopts a seven-stranded β-sandwich fold with one β sheet formed by four strands and the other by three strands. A disulfide bond between Cys25 and Cys80 links strands the two sheets in the folded state of the protein. β2m is vital for the correct folding, assembly, and cell-surface expression of the MHC-I complex. As part of normal cell turnover, β2m is released from the MHC-I complex and carried to the kidney where it is degraded. Upon renal failure, serum levels of β2m increase up to ~60 times above the normal level of 0.1 µM, and the protein aggregates into insoluble amyloid fibrils in the joints (2,3). Elevated β2m concentrations alone, however, are not sufficient to trigger fibrillogenesis (4,5). β2m amyloid formation must therefore result from features unique to hemodialysis, but the exact cause in vivo is not known. ®2m amyloid fibrils can be generated in vitro, though, under acidic conditions (pH < 3.6) (6), by removing the first six N-terminal amino acids (7), by mixing the protein with collagen at pH = 6.4 (8), by sonicating the protein in the presence of sodium dodecyl sulfate at pH = *Corresponding author Department of Chemistry, University of Massachusetts, Amherst, rwvachet@chem.umass.edu, Telephone: (413) 545-2733, Fax: (413) . † Department of Chemistry, Universidad Industrial de Santander, AA 678, Bucaramanga, Colombia SUPPORTING INFORMATION AVAILABLEAdditional data can be found in the Supporting Information. These data include (i) plots showing the intensities of unmodified and modified forms of fragment Ile1-Tyr10 for three trials in the absence of Cu; (ii) tables presenting the intensities of unmodified and modified forms of peptide fragments containing residues Asn83, Lys94, and Lys6; (iii) plots illustrating the intensities of unmodified and modified forms of fragment Ile1-Tyr10 in the absence of Cu and 2 hours after addition of Cu; (iv) plots illustrating the extent of NHSA modification of β2m res...

Abstractb-2-Microglobulin (b2m) is deposited as amyloid fibrils in the bones and joints of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that biologically relevant amounts of Cu(II) can cause b2m to be converted to amyloid fibrils under physiological conditions in vitro. In this work, dynamic light scattering, mass spectrometry, and size-exclusion chromatography are used to characterize the role that Cu plays in the formation of oligomeric intermediates that precede fibril formation. Cu(II) is found to be necessary for the stability of the dimer and an initial form of the tetramer. The initially formed tetramer then undergoes a structural change to a state that no longer binds Cu(II) before progressing to a hexameric state. Based on these results, we propose that the lag phase associated with b2m fibril formation is partially accounted for by the structural transition of the tetramer that results in Cu(II) loss. Consistent with this observation is the determination that the mature b2m amyloid fibrils do not contain Cu. Thus, Cu(II) appears to play a catalytic role by enabling the organization of the necessary oligomeric intermediates that precede b2m amyloid formation.

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9. Mol Cancer Res; 9(12); 1621-31. Ó2011 AACR.

Blood circulates through nearly every organ including tumors. Therefore, plasma is a logical source to search for tumor-derived proteins and peptides. The challenge with plasma is that it is a complex bodily fluid composed of high concentrations of normal host proteins that obscure identification of tumor-derived molecules. To simplify plasma, we examined a low molecular weight (LMW) fraction (plasma peptidome) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. In the plasma peptidome of patients with ductal adenocarcinoma of the pancreas (DAP), a prominent peptide was identified from the QSOX1 parent protein. This peptide is stable in whole blood over 24 h and was present in 16 of 23 DAP patients and 4 of 5 patients with intraductal papillary mucinous neoplasm (IPMN). QSOX1 peptides were never identified in the plasma peptidome from 42 normal healthy donors using the same methods. Immunohistochemical staining of DAP tissue sections with anti-QSOX1 antibody shows overexpression of QSOX1 in tumor but not in adjacent stroma or normal ducts. Three of four pancreas tumor cell lines also express QSOX1 protein by Western blot analysis. This is the first report of QSOX1 peptides in plasma from DAP patients and makes the rare connection between a peptide in plasma from cancer patients and overexpression of the parent protein in tumors.

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Insulin degludec is an ultra-long-acting insulin analogue that is increasingly being used in diabetes due to its favourable efficacy and safety profile. Thus, there is an increasing demand for a reliable and specific analytical method to quantify insulin degludec for research, pharmaceutical industry and clinical applications. We developed and validated an automated, high-throughput method for quantification of insulin degludec in human blood samples across the expected clinical range combining immunopurification with high-resolution mass spectrometry. Validation was performed according to the requirements of the US Food and Drug Administration. The method satisfyingly met the following parameters: lower limit of quantification (120 pM), linearity, accuracy (error < 5%), precision (CV < 7.7%), selectivity, carry-over, recovery (89.7–97.2%), stability and performance in the presence of other insulin analogues. The method was successfully applied to clinical samples of patients treated with insulin degludec showing a good correlation with the administered dose (r2 = 0.78). High usability of the method is supported by the small specimen volume, automated sample processing and short analysis time. In conclusion, this reliable, easy-to-use and specific mass spectrometric insulin degludec assay offers great promise to address the current unmet need for standardized insulin analytics in academic and industrial research.

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