| Autor: |
Nwosu AJ; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Misal SA; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Truong T; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Carson RH; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Webber KGI; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Axtell NB; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Liang Y; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Johnston SM; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Virgin KL; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Smith EG; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Thomas GV; Knight Cancer Center, Oregon Health & Science University, Portland, Oregon 97239, United States., Morgan T; Department of Pathology, Oregon Health & Science University, Portland, Oregon 97239, United States., Price JC; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States., Kelly RT; Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States. |
| Abstrakt: |
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. |
