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Background: The multifunctional glycoprotein clusterin has been associated with late-onset Alzheimer's disease (AD). Further investigation to define the role of clusterin in AD phenotypes would be aided by the development of techniques to quantify level, potential post-translational modifications, and isoforms of clusterin. We have developed a quantitative technique based on multiple reaction monitoring (MRM) mass spectrometry to measure clusterin in human postmortem brain tissues. Results: A stable isotope-labeled concatenated peptide (QconCAT) bearing selected peptides from clusterin was expressed with an in vitro translation system and purified. This clusterin QconCAT was validated for use as an internal standard for clusterin quantification using MRM mass spectrometry. Measurements were performed on the human postmortem frontal and temporal cortex from control and severe AD cases. During brain tissues processing, 1% SDS was used in the homogenization buffer to preserve potential post-translational modifications of clusterin. However, MRM quantifications in the brain did not suggest phosphorylation of Thr393, Ser394, and Ser396 residues reported for clusterin in serum. MRM quantifications in the frontal cortex demonstrated significantly higher (P < 0.01) level of clusterin in severe AD group (39.1 ± 9.1 pmol/mg tissue protein) in comparison to control group (25.4 ± 4.4 pmol/mg tissue protein). In the temporal cortex, the clusterin levels were not significantly different, 29.0 ± 7.9 pmol/mg tissue protein and 28.0 ± 8.4 pmol/mg tissue protein in control and severe AD groups, respectively. Conclusions: The proposed protocol is a universal quantitative technique to assess expression level of clusterin. It is expected that application of this protocol to quantification of various clusterin isoforms and potential post-translational modifications will be helpful in addressing the role of clusterin in AD. [ABSTRACT FROM AUTHOR]

Background: In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells., Method: Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry., Results: The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC., Conclusion: Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.