Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channel
Abstract Single‐cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic depth, throughput, and robustness, which we address here by a streamlined multiplexed workflow using data‐independent acquis...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Springer Nature
2023-09-01
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| Series: | Molecular Systems Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.15252/msb.202211503 |
| _version_ | 1797279736562450432 |
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| author | Marvin Thielert Ericka CM Itang Constantin Ammar Florian A Rosenberger Isabell Bludau Lisa Schweizer Thierry M Nordmann Patricia Skowronek Maria Wahle Wen‐Feng Zeng Xie‐Xuan Zhou Andreas‐David Brunner Sabrina Richter Mitchell P Levesque Fabian J Theis Martin Steger Matthias Mann |
| author_facet | Marvin Thielert Ericka CM Itang Constantin Ammar Florian A Rosenberger Isabell Bludau Lisa Schweizer Thierry M Nordmann Patricia Skowronek Maria Wahle Wen‐Feng Zeng Xie‐Xuan Zhou Andreas‐David Brunner Sabrina Richter Mitchell P Levesque Fabian J Theis Martin Steger Matthias Mann |
| author_sort | Marvin Thielert |
| collection | DOAJ |
| description | Abstract Single‐cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic depth, throughput, and robustness, which we address here by a streamlined multiplexed workflow using data‐independent acquisition (mDIA). We demonstrate automated and complete dimethyl labeling of bulk or single‐cell samples, without losing proteomic depth. Lys‐N digestion enables five‐plex quantification at MS1 and MS2 level. Because the multiplexed channels are quantitatively isolated from each other, mDIA accommodates a reference channel that does not interfere with the target channels. Our algorithm RefQuant takes advantage of this and confidently quantifies twice as many proteins per single cell compared to our previous work (Brunner et al, PMID 35226415), while our workflow currently allows routine analysis of 80 single cells per day. Finally, we combined mDIA with spatial proteomics to increase the throughput of Deep Visual Proteomics seven‐fold for microdissection and four‐fold for MS analysis. Applying this to primary cutaneous melanoma, we discovered proteomic signatures of cells within distinct tumor microenvironments, showcasing its potential for precision oncology. |
| first_indexed | 2024-03-07T16:30:18Z |
| format | Article |
| id | doaj.art-31e6f44353a34972902d11ba35f80b5e |
| institution | Directory Open Access Journal |
| issn | 1744-4292 |
| language | English |
| last_indexed | 2024-03-07T16:30:18Z |
| publishDate | 2023-09-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj.art-31e6f44353a34972902d11ba35f80b5e2024-03-03T10:44:17ZengSpringer NatureMolecular Systems Biology1744-42922023-09-01199n/an/a10.15252/msb.202211503Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channelMarvin Thielert0Ericka CM Itang1Constantin Ammar2Florian A Rosenberger3Isabell Bludau4Lisa Schweizer5Thierry M Nordmann6Patricia Skowronek7Maria Wahle8Wen‐Feng Zeng9Xie‐Xuan Zhou10Andreas‐David Brunner11Sabrina Richter12Mitchell P Levesque13Fabian J Theis14Martin Steger15Matthias Mann16Department of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyHelmholtz Zentrum München – German Research Center for Environmental Health Institute of Computational Biology Neuherberg GermanyDepartment of Dermatology University of Zurich, University of Zurich Hospital Zurich SwitzerlandHelmholtz Zentrum München – German Research Center for Environmental Health Institute of Computational Biology Neuherberg GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyAbstract Single‐cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic depth, throughput, and robustness, which we address here by a streamlined multiplexed workflow using data‐independent acquisition (mDIA). We demonstrate automated and complete dimethyl labeling of bulk or single‐cell samples, without losing proteomic depth. Lys‐N digestion enables five‐plex quantification at MS1 and MS2 level. Because the multiplexed channels are quantitatively isolated from each other, mDIA accommodates a reference channel that does not interfere with the target channels. Our algorithm RefQuant takes advantage of this and confidently quantifies twice as many proteins per single cell compared to our previous work (Brunner et al, PMID 35226415), while our workflow currently allows routine analysis of 80 single cells per day. Finally, we combined mDIA with spatial proteomics to increase the throughput of Deep Visual Proteomics seven‐fold for microdissection and four‐fold for MS analysis. Applying this to primary cutaneous melanoma, we discovered proteomic signatures of cells within distinct tumor microenvironments, showcasing its potential for precision oncology.https://doi.org/10.15252/msb.202211503DIAdimethyl labelingmultiplexingsingle cellsspatial proteomics |
| spellingShingle | Marvin Thielert Ericka CM Itang Constantin Ammar Florian A Rosenberger Isabell Bludau Lisa Schweizer Thierry M Nordmann Patricia Skowronek Maria Wahle Wen‐Feng Zeng Xie‐Xuan Zhou Andreas‐David Brunner Sabrina Richter Mitchell P Levesque Fabian J Theis Martin Steger Matthias Mann Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channel Molecular Systems Biology DIA dimethyl labeling multiplexing single cells spatial proteomics |
| title | Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channel |
| title_full | Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channel |
| title_fullStr | Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channel |
| title_full_unstemmed | Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channel |
| title_short | Robust dimethyl‐based multiplex‐DIA doubles single‐cell proteome depth via a reference channel |
| title_sort | robust dimethyl based multiplex dia doubles single cell proteome depth via a reference channel |
| topic | DIA dimethyl labeling multiplexing single cells spatial proteomics |
| url | https://doi.org/10.15252/msb.202211503 |
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