Abstract PS18-38: Comparative analysis of differentially abundant proteins quantified by LC-MS/MS between flash frozen and laser microdissected OCT-embedded breast tumor samples

Abstract

Background: Proteomic studies are typically conducted using flash-frozen (FF) samples utilizing tandem mass spectrometry. However, FF samples are comprised of multiple cell types, making it difficult to ascertain the proteomic profiles of specific cells. Conversely, OCT-embedded (Optimal Cutting Temperature compound) specimens can undergo laser microdissection (LMD) to capture and study specific cell types separately from the cell mixture. In the current study, we compared proteomic data obtained from FF and OCT samples to determine if samples that are stored and processed differently produce comparable results. Methods: Proteins were extracted from FF and OCT-embedded invasive breast tumors from 5 female patients. FF samples were lysed via homogenization (FF/HOM) while OCT-embedded specimens underwent LMD to collect only tumor cells (OCT/LMD-T) or both tumor and stromal cells (OCT/LMD-TS) followed by incubation at 37°C. Proteins were extracted using the illustra triplePrep kit and then trypsin-digested, TMT-labeled, and processed by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Proteins were identified and quantified with Proteome Discoverer v1.4 and comparative analyses performed to identify proteins that were significantly differentially expressed amongst the different processing methods. Results: Among 4,950 proteins consistently quantified across all samples, 216 and 171 proteins were significantly differentially expressed (adjusted p-value 1) between FF/HOM vs. OCT/LMD-T and FF/HOM vs. OCT/LMD-TS, respectively, with most proteins being more highly abundant in the FF/HOM samples. PCA and unsupervised hierarchical clustering analysis with these 216 and 171 proteins were able to distinguish FF/HOM from OCT/LMD-T and OCT/LMD-TS samples, respectively. Likewise, PCA analysis and unsupervised clustering analysis using the 402 and 60 significantly differentially enriched GO terms (adjusted p-value (BH) Citation Format: Lori A. Sturtz, Guisong Wang, Punit Shah, Richard Searfoss, Praveen-Kumar Raj-Kumar, Jeffrey A. Hooke, J. Leigh Fantacone-Campbell, Brenda Deyarmin, Mary Lou Cutler, Rangaprasad Sarangarajan, Niven R. Narain, Hai Hu, Michael A. Kiebish, Albert J. Kovatich, Craig D. Shriver. Comparative analysis of differentially abundant proteins quantified by LC-MS/MS between flash frozen and laser microdissected OCT-embedded breast tumor samples [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-38.