8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease

Abstract

An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of eight proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2- and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid (CSF) proteins from subjects undergoing intravenous Ig treatment for Alzheimer's disease. Results from this study identify a number of protein expression changes that occur in CSF after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.

Figure 1

A schematic depiction of the workflow for using 8-plex isobaric reagents in shotgun proteomics studies. The basic workflow is the same as used for 4-plex isobaric reagents.

Figure 2

MS/MS of 2708.26 m/z from carbonic anhydrase from the test mixture of proteins quantified using 8-plex isobaric reagents. A zoom of the reporter ion region demonstrates the use of the 113, 114, 115, 116, 117, 118, 119, and 121 m/z ions to quantify relative protein abundance. Ions at 112 and 120 m/z are immonium ions for arginine and phenylalanine, respectively.

Figure 3

Visualization tool example for albumin developed in the R environment. As discussed in detail in the text, 3A are average peptide expression changes for both subjects, 3B and 3D are protein average expression changes for subjects A and B, 3C and 3E are peptide expression changes observed for subjects A and B, 3F is graphical representation of peptide expression changes using a heatmap to indicate increases or decreases in expression where the horizontal axis of the figure shows the amino acid position along the length of the protein.

Figure 4

Visualization tool applied to observed expression changes for clusterin / apolipoprotein J. As discussed in detail in the text, 4A are average peptide expression changes for both subjects, 4B and 4D are protein average expression changes for subjects A and B, 4C and 4E are peptide expression changes observed for subjects A and B, 4F is graphical representation of peptide expression changes using a heatmap to indicate increases or decreases in expression where the horizontal axis of the figure shows the amino acid position along the length of the protein.

Figure 5

Western analysis of clusterin / apolipoprotein J using primary antibodies against N-terminal and C-terminal epitopes.