Optimized Proteome Reduction for Integrative Top–Down Proteomics
Integrative top–down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective and routine assessment of proteomes. Nonetheless, any such assessments also require a rigorous review of methodology to ensure the deepest possible quantitative proteome ana...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-03-01
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| Series: | Proteomes |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2227-7382/11/1/10 |
| _version_ | 1797609289529950208 |
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| author | Breyer Woodland Aleksandar Necakov Jens R. Coorssen |
| author_facet | Breyer Woodland Aleksandar Necakov Jens R. Coorssen |
| author_sort | Breyer Woodland |
| collection | DOAJ |
| description | Integrative top–down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective and routine assessment of proteomes. Nonetheless, any such assessments also require a rigorous review of methodology to ensure the deepest possible quantitative proteome analyses. Here, we establish an optimized general protocol for proteome extracts to improve the reduction of proteoforms and, thus, resolution in 2DE. Dithiothreitol (DTT), tributylphosphine (TBP), and 2-hydroxyethyldisulfide (HED), combined and alone, were tested in one-dimensional SDS-PAGE (1DE), prior to implementation into a full 2DE protocol. Prior to sample rehydration, reduction with 100 mM DTT + 5 mM TBP yielded increased spot counts, total signal, and spot circularity (i.e., decreased streaking) compared to other conditions and reduction protocols reported in the literature. The data indicate that many widely implemented reduction protocols are significantly ‘under-powered’ in terms of proteoform reduction and thus, limit the quality and depth of routine top–down proteomic analyses. |
| first_indexed | 2024-03-11T05:59:24Z |
| format | Article |
| id | doaj.art-9eee839c31454155b18b5535474c42af |
| institution | Directory Open Access Journal |
| issn | 2227-7382 |
| language | English |
| last_indexed | 2024-03-11T05:59:24Z |
| publishDate | 2023-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Proteomes |
| spelling | doaj.art-9eee839c31454155b18b5535474c42af2023-11-17T13:33:28ZengMDPI AGProteomes2227-73822023-03-011111010.3390/proteomes11010010Optimized Proteome Reduction for Integrative Top–Down ProteomicsBreyer Woodland0Aleksandar Necakov1Jens R. Coorssen2Department of Biological Sciences, Faculty of Mathematics and Science, Brock University, St. Catharines, ON L2S 3A1, CanadaDepartment of Biological Sciences, Faculty of Mathematics and Science, Brock University, St. Catharines, ON L2S 3A1, CanadaDepartment of Biological Sciences, Faculty of Mathematics and Science, Brock University, St. Catharines, ON L2S 3A1, CanadaIntegrative top–down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective and routine assessment of proteomes. Nonetheless, any such assessments also require a rigorous review of methodology to ensure the deepest possible quantitative proteome analyses. Here, we establish an optimized general protocol for proteome extracts to improve the reduction of proteoforms and, thus, resolution in 2DE. Dithiothreitol (DTT), tributylphosphine (TBP), and 2-hydroxyethyldisulfide (HED), combined and alone, were tested in one-dimensional SDS-PAGE (1DE), prior to implementation into a full 2DE protocol. Prior to sample rehydration, reduction with 100 mM DTT + 5 mM TBP yielded increased spot counts, total signal, and spot circularity (i.e., decreased streaking) compared to other conditions and reduction protocols reported in the literature. The data indicate that many widely implemented reduction protocols are significantly ‘under-powered’ in terms of proteoform reduction and thus, limit the quality and depth of routine top–down proteomic analyses.https://www.mdpi.com/2227-7382/11/1/10dithiothreitol (DTT)2-hydroxyethyl disulfide (HED)protein speciesproteoformreducing agenttributylphosphine (TBP) |
| spellingShingle | Breyer Woodland Aleksandar Necakov Jens R. Coorssen Optimized Proteome Reduction for Integrative Top–Down Proteomics Proteomes dithiothreitol (DTT) 2-hydroxyethyl disulfide (HED) protein species proteoform reducing agent tributylphosphine (TBP) |
| title | Optimized Proteome Reduction for Integrative Top–Down Proteomics |
| title_full | Optimized Proteome Reduction for Integrative Top–Down Proteomics |
| title_fullStr | Optimized Proteome Reduction for Integrative Top–Down Proteomics |
| title_full_unstemmed | Optimized Proteome Reduction for Integrative Top–Down Proteomics |
| title_short | Optimized Proteome Reduction for Integrative Top–Down Proteomics |
| title_sort | optimized proteome reduction for integrative top down proteomics |
| topic | dithiothreitol (DTT) 2-hydroxyethyl disulfide (HED) protein species proteoform reducing agent tributylphosphine (TBP) |
| url | https://www.mdpi.com/2227-7382/11/1/10 |
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