Mass spectrometry quantification of clusterin in the human brain

Abstract

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 Thr(393), Ser(394), and Ser(396) 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.

Figure 1

Design, expression, and characterization of clusterin QconCAT. (A). Clusterin QconCAT includes five Q peptides (shown in green) with 6-amino acid long flanking regions (shown in yellow) concatenated into the sequence with N-terminal Met and C-terminal His₆-tag. (B) Coomassie R250 stained clusterin QconCAT after 15% polyacrylamide gel separation. (C) Stable isotope incorporation into the clusterin. MRM spectra for two representative transitions per Q₁, Q₄, and Q₅ peptides are shown. The pair transitions for light (unlabeled) and heavy (labeled) form of each Q peptide are color coordinated. Isotope incorporation factor for clusterin QconCAT was calculated based on combined data for all three peptides and presented as mean ± SD.

Figure 2

Representative standard curves for Q₁, Q₄, and Q₅ peptides. The area ratio of heavy to light peaks for a selected transition was plotted versus suplemented clusterin QconCAT amount for each Q peptide. Transitions: Q₁-t3 is 697.35/1035.51 and 702.36/1045.52; Q₄-t1 is 644.82/602.30 and 649.83/612.30; Q₅-t1 is 772.06/507.80 and 774.74/511.80.