Delving into proteoforms with higher throughput
ProQuant™ was used to study apolipoprotein E (apoE), a polymorphic protein with key roles in longevity and Alzheimer’s disease. Our experiments testing different samples from in vitro and in vivo experiments identified a number of key peptides of interest throughout the apoE protein. These included a small number which were genotype-specific, while others were ubiquitous across genotypes but contained modifications including oxidation and citrullination.
To facilitate analysis of many more samples in a higher throughput approach, a multiple reaction monitoring (MRM) assay was developed to accurately quantify these peptides in samples from a large patient cohort, stored in 96 well plates. In total, the abundances of 15 peptides derived from apoE were measured in over 1000 human serum samples. Other apoE measures (e.g. an apoE/apoE4 ELISA) had been performed on these samples and the apoE genotypes of the donors determined.
The results were fascinating. PCA identified two key principal components. However, despite the thousands of publications investigating apoE genotype over the last few decades the primary principal component in our model (shown in blue circles in the graph) predominantly reflected the presence and absence of PTMs such as oxidation and citrullination. Genotype, which most would expect to be the determining factor for apoE function appears predominantly in PC2.