Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell Type
As the primary innate immune cells of the brain, microglia play a key role in various homeostatic and disease-related processes. To carry out their numerous functions, microglia adopt a wide range of phenotypic states. The proteomic landscape represents a more accurate molecular representation of th...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-11-01
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| Series: | Proteomes |
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| Online Access: | https://www.mdpi.com/2227-7382/12/4/35 |
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| author | Jessica Wohlfahrt Jennifer Guergues Stanley M. Stevens |
| author_facet | Jessica Wohlfahrt Jennifer Guergues Stanley M. Stevens |
| author_sort | Jessica Wohlfahrt |
| collection | DOAJ |
| description | As the primary innate immune cells of the brain, microglia play a key role in various homeostatic and disease-related processes. To carry out their numerous functions, microglia adopt a wide range of phenotypic states. The proteomic landscape represents a more accurate molecular representation of these phenotypes; however, microglia present unique challenges for proteomic analysis. This study implemented a streamlined liquid- and gas-phase fractionation method with data-dependent acquisition (DDA) and parallel accumulation–serial fragmentation (PASEF) analysis on a TIMS-TOF instrument to compile a comprehensive protein library obtained from adult-derived, immortalized mouse microglia with low starting material (10 µg). The empirical library consisted of 9140 microglial proteins and was utilized to identify an average of 7264 proteins/run from single-shot, data-independent acquisition (DIA)-based analysis microglial cell lysate digest (200 ng). Additionally, a predicted library facilitated the identification of 7519 average proteins/run from the same DIA data, revealing complementary coverage compared with the empirical library and collectively increasing coverage to approximately 8000 proteins. Importantly, several microglia-relevant pathways were uniquely identified with the empirical library approach. Overall, we report a simplified, reproducible approach to address the proteome complexity of microglia using low sample input and show the importance of library optimization for this phenotypically diverse cell type. |
| format | Article |
| id | doaj-art-debdce7b67504c6eacf1ecb71eaf4f0e |
| institution | Kabale University |
| issn | 2227-7382 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Proteomes |
| spelling | doaj-art-debdce7b67504c6eacf1ecb71eaf4f0e2024-12-27T14:49:28ZengMDPI AGProteomes2227-73822024-11-011243510.3390/proteomes12040035Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell TypeJessica Wohlfahrt0Jennifer Guergues1Stanley M. Stevens2Department of Molecular Biosciences, University of South Florida, Tampa, FL 33620, USADepartment of Molecular Biosciences, University of South Florida, Tampa, FL 33620, USADepartment of Molecular Biosciences, University of South Florida, Tampa, FL 33620, USAAs the primary innate immune cells of the brain, microglia play a key role in various homeostatic and disease-related processes. To carry out their numerous functions, microglia adopt a wide range of phenotypic states. The proteomic landscape represents a more accurate molecular representation of these phenotypes; however, microglia present unique challenges for proteomic analysis. This study implemented a streamlined liquid- and gas-phase fractionation method with data-dependent acquisition (DDA) and parallel accumulation–serial fragmentation (PASEF) analysis on a TIMS-TOF instrument to compile a comprehensive protein library obtained from adult-derived, immortalized mouse microglia with low starting material (10 µg). The empirical library consisted of 9140 microglial proteins and was utilized to identify an average of 7264 proteins/run from single-shot, data-independent acquisition (DIA)-based analysis microglial cell lysate digest (200 ng). Additionally, a predicted library facilitated the identification of 7519 average proteins/run from the same DIA data, revealing complementary coverage compared with the empirical library and collectively increasing coverage to approximately 8000 proteins. Importantly, several microglia-relevant pathways were uniquely identified with the empirical library approach. Overall, we report a simplified, reproducible approach to address the proteome complexity of microglia using low sample input and show the importance of library optimization for this phenotypically diverse cell type.https://www.mdpi.com/2227-7382/12/4/35microgliaDIA-PASEFDDA library generationdeep proteomicsDIA methods development |
| spellingShingle | Jessica Wohlfahrt Jennifer Guergues Stanley M. Stevens Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell Type Proteomes microglia DIA-PASEF DDA library generation deep proteomics DIA methods development |
| title | Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell Type |
| title_full | Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell Type |
| title_fullStr | Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell Type |
| title_full_unstemmed | Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell Type |
| title_short | Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell Type |
| title_sort | deep proteome coverage of microglia using a streamlined data independent acquisition based proteomic workflow method consideration for a phenotypically diverse cell type |
| topic | microglia DIA-PASEF DDA library generation deep proteomics DIA methods development |
| url | https://www.mdpi.com/2227-7382/12/4/35 |
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