Determining Allele-Specific Protein Expression (ASPE) Using a QconCAT-Based Proteomics Method: a Novel Approach to Identify Cis-acting Genetic Variants

Abstract

Cis-acting variants regulate gene expression in an allele-specific manner, and measuring allele-specific expression (ASE) is thus a powerful approach for identifying cis-regulatory genetic variants. Most ASE studies are conducted at the mRNA level; however, the correlation between mRNA and protein expressions is very poor for many genes, and genetic variants affecting expression at the post-transcriptional level cannot be determined by measuring mRNA ASE. In the present study, we developed a novel targeted proteomics approach for quantification of allele-specific protein expression (ASPE) based on scheduled high resolution multiple reaction monitoring (sMRM-HR) with a heavy stable isotope-labeled quantitative concatamer (QconCAT) internal protein standard. This strategy was applied to the determination of the ASPE of UGT2B15 in human livers using the common UGT2B15 nonsynonymous variant rs1902023 (i.e. Y85D) as the marker to differentiate expressions from the two alleles. The QconCAT standard contains both the wild type tryptic peptide and the Y85D mutant peptide at a ratio of 1:1 to ensure accurate measurement of the ASPE of UGT2B15. The results from 18 UGT2B15 Y85D heterozygotes revealed that the ratios between wild type Y allele and mutant D allele varied from 0.60 to 1.46, indicating the presence of cis-regulatory variants. In addition, we observed no significant correlations between the ASPE and mRNA ASE of UGT2B15, suggesting the involvement of different cis-acting variants in regulating the transcription and translation processes of the gene. This novel ASPE approach provides a powerful tool for capturing cis-genetic variants involved in posttranscription processes, an important yet understudied area of research.