Quantitative Analysis of Apolipoproteins in Human HDL by Top-Down Differential Mass Spectrometry

Abstract

The field of quantitative, label-free proteomics has evolved significantly over time, with most experiments performed “bottom-up” using proteolyzed protein mixtures. In these experiments, statistically significant peptide abundance differences between two or more experimental conditions are determined, and their corresponding proteins later identified. Recently, the rationale for extending this experimental design to mixtures of intact proteins has become clear, as analysis at the protein level allows for the independent detection of each protein form present, including those modified posttranslationally. This provides a level of specificity lost in bottom-up experiments. As such, the application of label-free top-down differential mass spectrometry has provided a means for understanding the subtle protein changes that define a particular phenotype. Described here is an approach for the top-down label-free quantitative analysis of the proteins which constitute human high-density lipoprotein particles. The methodology is conceptually very straightforward; however, it does require a level of rigor and consistency typically not addressed by more conventional proteomics experiments.