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
Authors: Sturtz Lori A. , Wang Guisong , Shah Punit , Searfoss Richard , Raj-Kumar Praveen-Kumar , Hooke Jeffrey A. , Fantacone-Campbell J. Leigh , Deyarmin Brenda , Cutler Mary Lou , Sarangarajan Rangaprasad , Narain Niven R. , Hu Hai , Kiebish Michael A. , Kovatich Albert J. , Shriver Craig D. 
Published: February 2021

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 &lt; 0.05; |log2 FC| &gt; 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) &lt; 0.2) in the FF/HOM vs. OCT/LMD-T and FF/HOM vs OCT/LMD-TS comparisons, respectively, not only distinguished OCT/LMD from FF/HOM samples but also separated LA and LB1 breast cancer subtypes within each storage/preparation method from one another. Although FF/HOM appears to be more similar to OCT/LMD-TS than OCT/LMD-T based on the number of differentially enriched proteins (216 vs. 171; p=0.022) and GO terms (402 vs. 60; p &lt; 2.2 x 10-16), FF/HOM shows no greater similarity to OCT/LMD-TS than OCT/LMD-T based on PCA analysis with either proteins or GO terms ( based on weighted distance for pairwise samples, p = 0.97 from paired t-test). No significantly differentially enriched proteins or GO terms were detected between the OCT/LMD-T and OCT/LMD-TS samples but trended differences were detected. Conclusions: The proteomic profiles of the OCT/LMD-TS samples were more similar to those from OCT/LMD-T samples than FF/HOM samples, suggesting a strong influence from the sample processing methods. These results indicate that in LC-MS/MS proteomic studies, FF/HOM samples exhibit different protein profiles from OCT/LMD samples and thus, results from these two different methods cannot be directly compared. Our study also provides preliminary data for designing new studies to explore why OCT/LMD-TS samples are more similar to OCT/LMD-T than to FF/HOM samples, and to separate LA from LB1 samples. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DOD or the Departments of the Army, Navy or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government.
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.