Optimisation of a Targeted Mass Spectrometry Assay to Investigate Nuclear Lamina Dynamics

Nuclear lamins are intermediate filament proteins within the nuclear membrane that are involved in a number of cellular processes such as providing structural support to the nucleus, transcriptional regulation and mechanosensing. They are also considered biomarkers in a number of different cancers. Here, a targeted multiple-reaction monitoring-based liquid chromatography-tandem mass spectrometry (MRM-LCMS/ MS) assay was developed and optimised to examine the protein expression of the four main mammalian nuclear lamin isoforms in biological samples of human and mouse origin. As part of this assay development, instrument optimisation was performed and the results suggested that optimisation of the collision energy (CE), fragmentor voltage (FV), and several electrospray ionisation source parameters can lead to profound improvements in assay sensitivity. This optimised MS method was then used to provide semi-quantitative analysis of the protein expression of nuclear lamin isoforms in cell lines and mouse models. Analysis of secondary cell lines was conducted to investigate the expression of nuclear lamins in cells representative of different tissue stiffnesses, in cell lines representative of different B-cell malignancies, including Burkitt lymphoma and multiple myeloma, and in a Burkitt lymphoma cell line devoid of activation-induced cytidine deaminase (AID), a key regulating enzyme involved in B-cell activation. Additionally, mouse models were also used to examine nuclear lamin expression in the lamin B1-overexpressing neurodegenerative disease autosomal dominant leukodystrophy (ADLD) and in knock-down mouse models of LMNB1 and ATG7. Use of the optimised LC-MRM-MS/MS method allowed for the limited detection and semi-quantitation of different nuclear lamin isoforms in these samples. The targeted nature of the assay allows for future work to focus on the seamless translation of this method into a fully quantitative assay to be implemented in the measurement of various nuclear lamin isoforms in clinical samples.