Figure 1. Multiplexed analysis of individual HDL proteomes from AAA patients: shotgun proteomics workflow.

(a) HDL particles were isolated by sequential ultracentrifugation. Protein extracts were loaded onto SDS-PAGE gels, and proteins were concentrated in the stacking gel. After in-gel trypsin digestion, peptides were labeled with 4-plex iTRAQ reagents and then analyzed by LC-MS/MS. (b) A total of 7 iTRAQ experiments were performed. In each experiment, samples from 2 AAA patients and 1 control subject were compared with an internal control. The internal control was a pool of all the individual samples used in the study. (c) The statistical model for the quantitative data decomposes the total technical variance into the spectral, peptide, and protein variance components and provides a general framework to fully integrate quantitative and error information, allowing a comparative analysis of the results obtained from the 7 iTRAQ experiments.