Quantification of the Lamin A/C Transcript Variants in Cancer Cell Lines by Targeted Absolute Quantitative Proteomics and Correlation with mRNA Expression

Abstract

<i>Lamin A/C</i> proteins have key roles in nuclear structural integrity and chromosomal stability. <i>Lamin A/C</i> cumulative protein expression of all variants is reported by semi-quantitative Western blotting. To date, there have not been specific antibodies for the individual <i>Lamin A/C</i> transcript variants. We developed a mass spectrometric approach for the quantification of <i>Lamin A/C</i> transcript variants. A signature peptide for each specific splice variant of <i>Lamin A/C</i> was selected. A LC-MS/MS assay based on the selected signature peptides and their labeled internal standards was established to measure the expression of <i>Lamin A/C</i> transcript variant concentrations. The method validation was carried out according to Food and Drug Administration (FDA) guidelines. The expression levels of the <i>Lamin A/C</i> transcript variants were measured in samples derived from MCF7 and U937 cell lines. RT-qPCR assay was also used to quantitate and compare the mRNA expression of splice variants of <i>Lamin A/C</i>. The established and validated method showed a great linearity, sensitivity, and precision. The different expressed <i>Lamin A/C</i> variants in different cell lines were measured and their levels were in concordance with qRT-PCR results. The developed method is reproducible, reliable, and sensitive for measuring different <i>Lamin A/C</i> transcript variants in different cell lines.