Automated sample preparation with SP3 for low‐input clinical proteomics
Abstract High‐throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including fresh‐frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single‐pot solid‐phase‐enhanced sample preparation (...
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| Format: | Article |
| Language: | English |
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Springer Nature
2020-01-01
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| Series: | Molecular Systems Biology |
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| Online Access: | https://doi.org/10.15252/msb.20199111 |
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| _version_ | 1849235640258920448 |
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| author | Torsten Müller Mathias Kalxdorf Rémi Longuespée Daniel N Kazdal Albrecht Stenzinger Jeroen Krijgsveld |
| author_facet | Torsten Müller Mathias Kalxdorf Rémi Longuespée Daniel N Kazdal Albrecht Stenzinger Jeroen Krijgsveld |
| author_sort | Torsten Müller |
| collection | DOAJ |
| description | Abstract High‐throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including fresh‐frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single‐pot solid‐phase‐enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96‐well format. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands‐on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low‐input samples, reproducibly quantifying 500–1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their separation into known histological growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end‐to‐end sample preparation and LCMS analysis of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost‐effective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clinical and non‐clinical applications. |
| format | Article |
| id | doaj-art-bbd2898a56b34226bafb9ba7a5f5aeda |
| institution | Kabale University |
| issn | 1744-4292 |
| language | English |
| publishDate | 2020-01-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj-art-bbd2898a56b34226bafb9ba7a5f5aeda2025-08-20T04:02:44ZengSpringer NatureMolecular Systems Biology1744-42922020-01-0116111910.15252/msb.20199111Automated sample preparation with SP3 for low‐input clinical proteomicsTorsten Müller0Mathias Kalxdorf1Rémi Longuespée2Daniel N Kazdal3Albrecht Stenzinger4Jeroen Krijgsveld5German Cancer Research Center (DKFZ)German Cancer Research Center (DKFZ)Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg UniversityInstitute of Pathology, Heidelberg UniversityInstitute of Pathology, Heidelberg UniversityGerman Cancer Research Center (DKFZ)Abstract High‐throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including fresh‐frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single‐pot solid‐phase‐enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96‐well format. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands‐on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low‐input samples, reproducibly quantifying 500–1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their separation into known histological growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end‐to‐end sample preparation and LCMS analysis of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost‐effective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clinical and non‐clinical applications.https://doi.org/10.15252/msb.20199111automationclinical proteomicsFFPElow‐inputSP3 |
| spellingShingle | Torsten Müller Mathias Kalxdorf Rémi Longuespée Daniel N Kazdal Albrecht Stenzinger Jeroen Krijgsveld Automated sample preparation with SP3 for low‐input clinical proteomics Molecular Systems Biology automation clinical proteomics FFPE low‐input SP3 |
| title | Automated sample preparation with SP3 for low‐input clinical proteomics |
| title_full | Automated sample preparation with SP3 for low‐input clinical proteomics |
| title_fullStr | Automated sample preparation with SP3 for low‐input clinical proteomics |
| title_full_unstemmed | Automated sample preparation with SP3 for low‐input clinical proteomics |
| title_short | Automated sample preparation with SP3 for low‐input clinical proteomics |
| title_sort | automated sample preparation with sp3 for low input clinical proteomics |
| topic | automation clinical proteomics FFPE low‐input SP3 |
| url | https://doi.org/10.15252/msb.20199111 |
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