Mapping Nanoscale‐To‐Single‐Cell Phosphoproteomic Landscape by Chip‐DIA
Abstract Protein phosphorylation plays a crucial role in regulating disease phenotypes and serves as a key target for drug development. Mapping nanoscale‐to‐single‐cell samples can unravel the heterogeneity of cellular signaling events. However, it remains a formidable analytical challenge due to th...
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| Format: | Article |
| Language: | English |
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Wiley
2025-01-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.202402421 |
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| author | Gul Muneer Sofani Tafesse Gebreyesus Ciao‐Syuan Chen Tzu‐Tsung Lee Fengchao Yu Chih‐An Lin Min‐Shu Hsieh Alexey I. Nesvizhskii Chao‐Chi Ho Sung‐Liang Yu Hsiung‐Lin Tu Yu‐Ju Chen |
| author_facet | Gul Muneer Sofani Tafesse Gebreyesus Ciao‐Syuan Chen Tzu‐Tsung Lee Fengchao Yu Chih‐An Lin Min‐Shu Hsieh Alexey I. Nesvizhskii Chao‐Chi Ho Sung‐Liang Yu Hsiung‐Lin Tu Yu‐Ju Chen |
| author_sort | Gul Muneer |
| collection | DOAJ |
| description | Abstract Protein phosphorylation plays a crucial role in regulating disease phenotypes and serves as a key target for drug development. Mapping nanoscale‐to‐single‐cell samples can unravel the heterogeneity of cellular signaling events. However, it remains a formidable analytical challenge due to the low detectability, abundance, and stoichiometry of phosphorylation sites. Here, we present a Chip‐DIA strategy, integrating a microfluidic‐based phosphoproteomic chip (iPhosChip) with data‐independent acquisition mass spectrometry (DIA‐MS) for ultrasensitive nanoscale‐to‐single‐cell phosphoproteomic profiling. The iPhosChip operates as an all‐in‐one station that accommodates both quantifiable cell capture/imaging and the entire phosphoproteomic workflow in a highly streamlined and multiplexed manner. Coupled with a sample size‐comparable library‐based DIA‐MS strategy, Chip‐DIA achieved ultra‐high sensitivity, detecting 1076±158 to 15869±1898 phosphopeptides from 10±0 to 1013±4 cells, and revealed the first single‐cell phosphoproteomic landscape comprising druggable sites and basal phosphorylation‐mediated networks in lung cancer. Notably, the sensitivity and coverage enabled the illumination of heterogeneous cytoskeleton remodeling and cytokeratin signatures in patient‐derived cells resistant to third‐generation EGFR therapy, stratifying mixed‐lineage adenocarcinoma‐squamous cell carcinoma subtypes, and identifying alternative targeted therapy for late‐stage patients. With flexibility in module design and functionalization, Chip‐DIA can be adapted to other PTM‐omics to explore dysregulated PTM landscapes, thereby guiding therapeutic strategies toward precision oncology. |
| format | Article |
| id | doaj-art-d8e21484a4a4423196bd8d05457c08e0 |
| institution | DOAJ |
| issn | 2198-3844 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advanced Science |
| spelling | doaj-art-d8e21484a4a4423196bd8d05457c08e02025-08-20T02:47:33ZengWileyAdvanced Science2198-38442025-01-01121n/an/a10.1002/advs.202402421Mapping Nanoscale‐To‐Single‐Cell Phosphoproteomic Landscape by Chip‐DIAGul Muneer0Sofani Tafesse Gebreyesus1Ciao‐Syuan Chen2Tzu‐Tsung Lee3Fengchao Yu4Chih‐An Lin5Min‐Shu Hsieh6Alexey I. Nesvizhskii7Chao‐Chi Ho8Sung‐Liang Yu9Hsiung‐Lin Tu10Yu‐Ju Chen11Institute of Chemistry Academia Sinica Taipei 115201 TaiwanInstitute of Chemistry Academia Sinica Taipei 115201 TaiwanInstitute of Chemistry Academia Sinica Taipei 115201 TaiwanInstitute of Chemistry Academia Sinica Taipei 115201 TaiwanDepartment of Pathology University of Michigan Ann Arbor MI 48109 USADepartment of Internal Medicine National Taiwan University Hospital Taipei 10051 TaiwanDepartment of Pathology National Taiwan University Cancer Center Taipei 10617 TaiwanDepartment of Pathology University of Michigan Ann Arbor MI 48109 USADepartment of Internal Medicine National Taiwan University Hospital Taipei 10051 TaiwanDepartment of Clinical Laboratory Science and Medical Biotechnology College of Medicine National Taiwan University Taipei 10048 TaiwanInstitute of Chemistry Academia Sinica Taipei 115201 TaiwanInstitute of Chemistry Academia Sinica Taipei 115201 TaiwanAbstract Protein phosphorylation plays a crucial role in regulating disease phenotypes and serves as a key target for drug development. Mapping nanoscale‐to‐single‐cell samples can unravel the heterogeneity of cellular signaling events. However, it remains a formidable analytical challenge due to the low detectability, abundance, and stoichiometry of phosphorylation sites. Here, we present a Chip‐DIA strategy, integrating a microfluidic‐based phosphoproteomic chip (iPhosChip) with data‐independent acquisition mass spectrometry (DIA‐MS) for ultrasensitive nanoscale‐to‐single‐cell phosphoproteomic profiling. The iPhosChip operates as an all‐in‐one station that accommodates both quantifiable cell capture/imaging and the entire phosphoproteomic workflow in a highly streamlined and multiplexed manner. Coupled with a sample size‐comparable library‐based DIA‐MS strategy, Chip‐DIA achieved ultra‐high sensitivity, detecting 1076±158 to 15869±1898 phosphopeptides from 10±0 to 1013±4 cells, and revealed the first single‐cell phosphoproteomic landscape comprising druggable sites and basal phosphorylation‐mediated networks in lung cancer. Notably, the sensitivity and coverage enabled the illumination of heterogeneous cytoskeleton remodeling and cytokeratin signatures in patient‐derived cells resistant to third‐generation EGFR therapy, stratifying mixed‐lineage adenocarcinoma‐squamous cell carcinoma subtypes, and identifying alternative targeted therapy for late‐stage patients. With flexibility in module design and functionalization, Chip‐DIA can be adapted to other PTM‐omics to explore dysregulated PTM landscapes, thereby guiding therapeutic strategies toward precision oncology.https://doi.org/10.1002/advs.202402421library DIAlung cancermicrofluidicsphosphorylationsingle‐cell phosphoproteomics |
| spellingShingle | Gul Muneer Sofani Tafesse Gebreyesus Ciao‐Syuan Chen Tzu‐Tsung Lee Fengchao Yu Chih‐An Lin Min‐Shu Hsieh Alexey I. Nesvizhskii Chao‐Chi Ho Sung‐Liang Yu Hsiung‐Lin Tu Yu‐Ju Chen Mapping Nanoscale‐To‐Single‐Cell Phosphoproteomic Landscape by Chip‐DIA Advanced Science library DIA lung cancer microfluidics phosphorylation single‐cell phosphoproteomics |
| title | Mapping Nanoscale‐To‐Single‐Cell Phosphoproteomic Landscape by Chip‐DIA |
| title_full | Mapping Nanoscale‐To‐Single‐Cell Phosphoproteomic Landscape by Chip‐DIA |
| title_fullStr | Mapping Nanoscale‐To‐Single‐Cell Phosphoproteomic Landscape by Chip‐DIA |
| title_full_unstemmed | Mapping Nanoscale‐To‐Single‐Cell Phosphoproteomic Landscape by Chip‐DIA |
| title_short | Mapping Nanoscale‐To‐Single‐Cell Phosphoproteomic Landscape by Chip‐DIA |
| title_sort | mapping nanoscale to single cell phosphoproteomic landscape by chip dia |
| topic | library DIA lung cancer microfluidics phosphorylation single‐cell phosphoproteomics |
| url | https://doi.org/10.1002/advs.202402421 |
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