Background
There is great interest in detecting, characterizing and quantifying transactive response DNA binding protein of 43 kDa (TDP-43), and its post-translational modifications, due to its association with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis. Unfortunately, detailed analysis of TDP-43 in human biological matrices by immunometric
Conclusions
The aptamer-enrichment-HPLC-MS/MS method enabled highly selective quantification, enhanced sequence coverage and structural characterization of endogenous TDP-43.
Methods
Compared to immunoenrichment, aptamer-enrichment yielded cleaner recoveries of TDP-43. The aptamer-enrichment-HPLC-MS/MS method, compared to our previous method without enrichment, increased analytical sensitivity by 8.7-fold and 11.8-fold for endogenous TDP-43 in human cells and brain tissue, respectively. Critically, inclusion of the aptamer enrichment step improved sequence resolution and enabled identification of TDP-43 C-terminal fragments. Conclusions: The aptamer-enrichment-HPLC-MS/MS method enabled highly selective quantification, enhanced sequence coverage and structural characterization of endogenous TDP-43.
Results
The TDP-43 aptamer-enrichment-HPLC-MS/MS assay was linear from 0.37 to 2.55nmol/L, a range suitable for analysis of both human cells and brain tissue homogenates, and had a total CV of 14.8%. Quantitative TDP-43 peptide profiles were developed for cases of FTD with TDP-43 pathology and cases with no neurodegenerative pathology. Comparison with existing methods: Compared to immunoenrichment, aptamer-enrichment yielded cleaner recoveries of TDP-43. The aptamer-enrichment-HPLC-MS/MS method, compared to our previous method without enrichment, increased analytical sensitivity by 8.7-fold and 11.8-fold for endogenous TDP-43 in human cells and brain tissue, respectively. Critically, inclusion of the aptamer enrichment step improved sequence resolution and enabled identification of TDP-43 C-terminal fragments. Conclusions: The aptamer-enrichment-HPLC-MS/MS method enabled highly selective quantification, enhanced sequence coverage and structural characterization of endogenous TDP-43.