Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform
| Autor: | Rui Zhao, Naomi Uwugiaren, Ronald J. Moore, Mowei Zhou, Sarah M. Williams, Ying Zhu, Ljiljana Paša-Tolić, Irena Dapic, David R. Goodlett |
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| Rok vydání: | 2020 |
| Předmět: |
Proteomics
Surface Properties Sample processing Microfluidics 010402 general chemistry Top-down proteomics 01 natural sciences Analytical Chemistry Protein purification Tumor Cells Cultured Humans Urea Sample preparation Particle Size Maltose Myristoylation Chemistry 010401 analytical chemistry Microfluidic Analytical Techniques Neoplasm Proteins 0104 chemical sciences Biochemistry Acetylation Nanoparticles HeLa Cells |
| Zdroj: | Analytical Chemistry. 92:7087-7095 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.0c00467 |
| Popis: | 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 a 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 proteomic workflow. A unique combination of a nonionic detergent dodecyl-β-d-maltopyranoside (DDM) with urea as protein extraction buffer significantly improved both protein extraction efficiency and sample recovery. We hypothesize that the DDM detergent improves protein recovery by efficiently reducing nonspecific 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 research, particularly where clinical specimens are not available in amounts amenable to standard workflows. |
| Databáze: | OpenAIRE |
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