In-Depth Mass Spectrometry-Based Proteomics of Formalin-Fixed, Paraffin-Embedded Tissues with a Spatial Resolution of 50–200 μm
- Andikan J. NwosuAndikan J. NwosuDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Andikan J. Nwosu
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- Santosh A. MisalSantosh A. MisalDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Santosh A. Misal
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- Thy TruongThy TruongDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Thy Truong
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- Richard H. CarsonRichard H. CarsonDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Richard H. Carson
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- Kei G. I. WebberKei G. I. WebberDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Kei G. I. Webber
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- Nathaniel B. AxtellNathaniel B. AxtellDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Nathaniel B. Axtell
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- Yiran LiangYiran LiangDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Yiran Liang
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- S. Madisyn JohnstonS. Madisyn JohnstonDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by S. Madisyn Johnston
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- Kenneth L. VirginKenneth L. VirginDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Kenneth L. Virgin
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- Ethan G. SmithEthan G. SmithDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Ethan G. Smith
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- George V. ThomasGeorge V. ThomasKnight Cancer Center, Oregon Health & Science University, Portland, Oregon 97239, United StatesMore by George V. Thomas
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- Terry MorganTerry MorganDepartment of Pathology, Oregon Health & Science University, Portland, Oregon 97239, United StatesMore by Terry Morgan
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- John C. PriceJohn C. PriceDepartment of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by John C. Price
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- Ryan T. Kelly*Ryan T. Kelly*Email: [email protected]Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United StatesMore by Ryan T. Kelly
Abstract
Formalin-fixed, paraffin-embedded (FFPE) tissues are banked in large repositories to cost-effectively preserve valuable specimens for later study. With the rapid growth of spatial proteomics, FFPE tissues can serve as a more accessible alternative to more commonly used frozen tissues. However, extracting proteins from FFPE tissues is challenging due to cross-links formed between proteins and formaldehyde. Here, we have adapted the nanoPOTS sample processing workflow, which was previously applied to single cells and fresh-frozen tissues, to profile protein expression from FFPE tissues. Following the optimization of extraction solvents, times, and temperatures, we identified an average of 1312 and 3184 high-confidence master proteins from 10 μm thick FFPE-preserved mouse liver tissue squares having lateral dimensions of 50 and 200 μm, respectively. The observed proteome coverage for FFPE tissues was on average 88% of that achieved for similar fresh-frozen tissues. We also characterized the performance of our fully automated sample preparation and analysis workflow, termed autoPOTS, for FFPE spatial proteomics. This modified nanodroplet processing in one pot for trace samples (nanoPOTS) and fully automated processing in one pot for trace sample (autoPOTS) workflows provides the greatest coverage reported to date for high-resolution spatial proteomics applied to FFPE tissues. Data are available via ProteomeXchange with identifier PXD029729.
Cited By
This article is cited by 3 publications.
- Ximena Sanchez-Avila, Thy Truong, Xiaofeng Xie, Kei G. I. Webber, S. Madisyn Johnston, Hsien-Jung L. Lin, Nathaniel B. Axtell, Veronica Puig-Sanvicens, Ryan T. Kelly. Easy and Accessible Workflow for Label-Free Single-Cell Proteomics. Journal of the American Society for Mass Spectrometry 2023, Article ASAP.
- S. Madisyn Johnston, Kei G. I. Webber, Xiaofeng Xie, Thy Truong, Alissia Nydegger, Hsien-Jung L. Lin, Andikan Nwosu, Ying Zhu, Ryan T. Kelly. Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics. Journal of the American Society for Mass Spectrometry 2023, 34 (8) , 1701-1707. https://doi.org/10.1021/jasms.3c00159
- Dong-Kyu Lee, Stanislav S. Rubakhin, Jonathan V. Sweedler. Chemical Decrosslinking-Based Peptide Characterization of Formaldehyde-Fixed Rat Pancreas Using Fluorescence-Guided Single-Cell Mass Spectrometry. Analytical Chemistry 2023, 95 (16) , 6732-6739. https://doi.org/10.1021/acs.analchem.3c00612