A fully automated FAIMS-DIA mass spectrometry-based proteomic pipeline
Summary: Here, we present a standardized, “off-the-shelf” proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high throughput and scalability. This integrated pipeline streamlines a fully automated sample preparation platform, a data-independent...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-10-01
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| Series: | Cell Reports: Methods |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S266723752300245X |
| _version_ | 1827783615934627840 |
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| author | Luke Reilly Erika Lara Daniel Ramos Ziyi Li Caroline B. Pantazis Julia Stadler Marianita Santiana Jessica Roberts Faraz Faghri Ying Hao Mike A. Nalls Priyanka Narayan Yansheng Liu Andrew B. Singleton Mark R. Cookson Michael E. Ward Yue A. Qi |
| author_facet | Luke Reilly Erika Lara Daniel Ramos Ziyi Li Caroline B. Pantazis Julia Stadler Marianita Santiana Jessica Roberts Faraz Faghri Ying Hao Mike A. Nalls Priyanka Narayan Yansheng Liu Andrew B. Singleton Mark R. Cookson Michael E. Ward Yue A. Qi |
| author_sort | Luke Reilly |
| collection | DOAJ |
| description | Summary: Here, we present a standardized, “off-the-shelf” proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high throughput and scalability. This integrated pipeline streamlines a fully automated sample preparation platform, a data-independent acquisition (DIA) coupled with high-field asymmetric waveform ion mobility spectrometer (FAIMS) interface, and an optimized library-free DIA database search strategy. Our systematic evaluation of FAIMS-DIA showing single compensation voltage (CV) at −35 V not only yields the deepest proteome coverage but also best correlates with DIA without FAIMS. Our in-depth comparison of direct-DIA database search engines shows that Spectronaut outperforms others, providing the highest quantifiable proteins. Next, we apply three common DIA strategies in characterizing human induced pluripotent stem cell (iPSC)-derived neurons and show single-shot mass spectrometry (MS) using single-CV (−35 V)-FAIMS-DIA results in >9,000 quantifiable proteins with <10% missing values, as well as superior reproducibility and accuracy compared with other existing DIA methods. Motivation: Although mass spectrometry (MS)-based quantitative proteomics has been significantly advanced in the past decade, reproducibility and robustness are roadblocks preventing this technique from being widely applied to large biomedical research communities. We aim to streamline and automate major components of MS-based proteomics, providing an extensively optimized and validated platform for large-scale studies in modern proteomic laboratories and contract research organizations. |
| first_indexed | 2024-03-11T15:49:56Z |
| format | Article |
| id | doaj.art-70e5f58e885a4fae9da3862e8bf66e4e |
| institution | Directory Open Access Journal |
| issn | 2667-2375 |
| language | English |
| last_indexed | 2024-03-11T15:49:56Z |
| publishDate | 2023-10-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports: Methods |
| spelling | doaj.art-70e5f58e885a4fae9da3862e8bf66e4e2023-10-26T04:18:31ZengElsevierCell Reports: Methods2667-23752023-10-01310100593A fully automated FAIMS-DIA mass spectrometry-based proteomic pipelineLuke Reilly0Erika Lara1Daniel Ramos2Ziyi Li3Caroline B. Pantazis4Julia Stadler5Marianita Santiana6Jessica Roberts7Faraz Faghri8Ying Hao9Mike A. Nalls10Priyanka Narayan11Yansheng Liu12Andrew B. Singleton13Mark R. Cookson14Michael E. Ward15Yue A. Qi16Center for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Data Tecnica International, LLC, Glen Echo, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA; Data Tecnica International, LLC, Glen Echo, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA; Data Tecnica International, LLC, Glen Echo, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20814, USADepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USACenter for Alzheimer’s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; Corresponding authorSummary: Here, we present a standardized, “off-the-shelf” proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high throughput and scalability. This integrated pipeline streamlines a fully automated sample preparation platform, a data-independent acquisition (DIA) coupled with high-field asymmetric waveform ion mobility spectrometer (FAIMS) interface, and an optimized library-free DIA database search strategy. Our systematic evaluation of FAIMS-DIA showing single compensation voltage (CV) at −35 V not only yields the deepest proteome coverage but also best correlates with DIA without FAIMS. Our in-depth comparison of direct-DIA database search engines shows that Spectronaut outperforms others, providing the highest quantifiable proteins. Next, we apply three common DIA strategies in characterizing human induced pluripotent stem cell (iPSC)-derived neurons and show single-shot mass spectrometry (MS) using single-CV (−35 V)-FAIMS-DIA results in >9,000 quantifiable proteins with <10% missing values, as well as superior reproducibility and accuracy compared with other existing DIA methods. Motivation: Although mass spectrometry (MS)-based quantitative proteomics has been significantly advanced in the past decade, reproducibility and robustness are roadblocks preventing this technique from being widely applied to large biomedical research communities. We aim to streamline and automate major components of MS-based proteomics, providing an extensively optimized and validated platform for large-scale studies in modern proteomic laboratories and contract research organizations.http://www.sciencedirect.com/science/article/pii/S266723752300245XCP: BiotechnologyCP: Systems biology |
| spellingShingle | Luke Reilly Erika Lara Daniel Ramos Ziyi Li Caroline B. Pantazis Julia Stadler Marianita Santiana Jessica Roberts Faraz Faghri Ying Hao Mike A. Nalls Priyanka Narayan Yansheng Liu Andrew B. Singleton Mark R. Cookson Michael E. Ward Yue A. Qi A fully automated FAIMS-DIA mass spectrometry-based proteomic pipeline Cell Reports: Methods CP: Biotechnology CP: Systems biology |
| title | A fully automated FAIMS-DIA mass spectrometry-based proteomic pipeline |
| title_full | A fully automated FAIMS-DIA mass spectrometry-based proteomic pipeline |
| title_fullStr | A fully automated FAIMS-DIA mass spectrometry-based proteomic pipeline |
| title_full_unstemmed | A fully automated FAIMS-DIA mass spectrometry-based proteomic pipeline |
| title_short | A fully automated FAIMS-DIA mass spectrometry-based proteomic pipeline |
| title_sort | fully automated faims dia mass spectrometry based proteomic pipeline |
| topic | CP: Biotechnology CP: Systems biology |
| url | http://www.sciencedirect.com/science/article/pii/S266723752300245X |
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