Top-down proteomics is a powerful tool for characterizing genetic variations and post-translational modifications at intact protein level. However, one significant technical gap of top-down proteomics is the inability to analyze low amount of biological samples, which limits its access to isolated rare cells, fine-needle aspiration biopsies, and tissue substructures. Herein, we developed an ultrasensitive top-down platform by incorporating a microfluidic sample preparation system, termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples), into a top-down proteomics workflow. A unique combination of a nonionic detergent dodecyl-ß-D-maltopyranoside (DDM) with urea as protein extraction buffer significantly im-proved both protein extraction efficiency and sample recovery. We hypothesize that the DDM detergent improves protein recovery by efficiently reducing non-specific adsorption of intact proteins on container surfaces, while urea serves as a strong denaturant to disrupt noncovalent complexes and release intact proteins for downstream analysis. The nanoPOTS-based top-down platform reproducibly and quantitatively identified ~170 to ~620 proteoforms from ~70 to ~770 HeLa cells containing ~10 to ~115 ng of total protein. A variety of post-translational modifications including acetylation, myristoylation, and iron binding were identified using only less than 800 cells. We anticipate the nanoPOTS top-down proteomics platform will be broadly applicable in biomedical researches, particularly where clinical specimens are not available in amounts amenable to standard workflows.
Revised: July 10, 2020 |
Published: May 19, 2020
Zhou M., N.O. Uwugiaren, S.M. Williams, R.J. Moore, R. Zhao, D. Goodlett, and I. Dapic, et al. 2020.Sensitive Top-Down Proteomics Analysis of Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform.Analytical Chemistry 92, no. 10:7087–7095.PNNL-SA-149823.doi:10.1021/acs.analchem.0c00467