摘要:PS18-38:快速冷冻和激光显微解剖oct包埋乳腺肿瘤样品LC-MS/MS定量差异丰富蛋白的比较分析

Lori A. Sturtz, Guisong Wang, Punit Shah, R. Searfoss, Praveen-Kumar Raj-Kumar, J. Hooke, J. Fantacone-Campbell, B. Deyarmin, M. Cutler, R. Sarangarajan, N. Narain, Hai Hu, M. Kiebish, A. Kovatich, C. Shriver
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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. 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引用次数: 0

摘要

背景:蛋白质组学研究通常使用快速冷冻(FF)样品进行串联质谱分析。然而,FF样品由多种细胞类型组成,因此很难确定特定细胞的蛋白质组学特征。相反,oct包埋(最佳切割温度化合物)标本可以进行激光显微解剖(LMD),以从细胞混合物中单独捕获和研究特定的细胞类型。在目前的研究中,我们比较了从FF和OCT样品中获得的蛋白质组学数据,以确定存储和处理方式不同的样品是否产生可比较的结果。方法:分别从5例女性乳腺浸润性肿瘤FF和oct中提取蛋白。FF样品通过均质化(FF/HOM)裂解,OCT包埋标本进行LMD,仅收集肿瘤细胞(OCT/LMD- t)或肿瘤和基质细胞(OCT/LMD- ts),然后在37℃孵育。采用illustra triplePrep试剂盒提取蛋白质,胰蛋白酶消化,tmt标记,二维液相色谱-串联质谱(2D LC-MS/MS)处理。使用Proteome Discoverer v1.4对蛋白质进行鉴定和定量,并进行比较分析,以确定在不同处理方法中表达显著差异的蛋白质。结果:在所有样品中一致定量的4950种蛋白质中,有216种和171种蛋白质在FF/HOM与OCT/LMD-T和FF/HOM与OCT/LMD-TS之间的表达差异显著(调整p值1),其中大多数蛋白质在FF/HOM样品中含量更高。用这216个和171个蛋白进行PCA和无监督分层聚类分析,可以分别将FF/HOM与OCT/LMD-T和OCT/LMD-TS样品区分开来。同样,PCA分析和无监督聚类分析使用402和60显著差异富集的GO项(调整p值(BH)引用格式: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。快闪冷冻与激光显微解剖oct包埋乳腺肿瘤标本LC-MS/MS定量蛋白差异分析[摘要]。参见:2020年圣安东尼奥乳腺癌虚拟研讨会论文集;2020年12月8-11日;费城(PA): AACR;癌症杂志,2021;81(4增刊):摘要nr PS18-38。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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
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.
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