Proteomic studies are typically conducted using flash-frozen (FF) samples utilizing tandem mass spectrometry (MS). However, FF specimens 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. Proteins were extracted from FF and OCT-embedded invasive breast tumors from 5 female patients. FF specimens 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. Among the 4,950 proteins consistently quantified across all samples, 216 and 171 proteins were significantly differentially expressed (adjusted p-value < 0.05; |log2 FC|> 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. Similar analyses using significantly differentially enriched GO terms also discriminated FF/HOM from OCT/LMD samples. No significantly differentially expressed proteins were detected between the OCT/LMD-T and OCT/LMD-TS samples but trended differences were detected. 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 expression profiles from OCT/LMD samples and thus, results from these two different methods cannot be directly compared.

中文翻译：

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通过LC-MS / MS定量分析的快速冷冻和激光显微切割OCT包埋的乳腺肿瘤样品之间差异丰富的蛋白质的比较分析

蛋白质组学研究通常使用串联质谱（MS）使用急冻（FF）样品进行。但是，FF标本由多种细胞类型组成，因此难以确定特定细胞的蛋白质组学特征。相反，可以将嵌入OCT的（最佳切割温度化合物）标本进行激光显微切割（LMD），以从细胞混合物中分别捕获和研究特定的细胞类型。在当前的研究中，我们比较了从FF和OCT样品获得的蛋白质组数据，以确定存储和处理的样品是否产生可比的结果。从5名女性患者的FF和OCT浸润性浸润性乳腺癌中提取蛋白质。通过匀浆（FF / HOM）裂解FF标本，对OCT包埋的标本进行LMD收集仅肿瘤细胞（OCT / LMD-T）或肿瘤和基质细胞（OCT / LMD-TS），然后在37°C孵育。使用illustra TriplePrep试剂盒提取蛋白质，然后用​​胰蛋白酶消化，TMT标记并通过二维液相色谱-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，并进行比较分析以鉴定在不同加工方法之间差异显着的蛋白质。在所有样品中一致定量的4,950种蛋白质中，FF / HOM与OCT / LMD-T和FF / HOM与FF / HOM相比，有216和171种蛋白质显着差异表达（校正后的p值<0.05； | log2 FC |> 1）。 OCT / LMD-TS，分别是，大多数蛋白质在FF / HOM样品中含量更高。PCA和使用这216和171蛋白的无监督分层聚类分析能够分别将FF / HOM与OCT / LMD-T和OCT / LMD-TS样品区分开。使用显着差异富集的GO项进行的类似分析也将FF / HOM与OCT / LMD样品区分开。在OCT / LMD-T和OCT / LMD-TS样品之间未检测到显着差异表达的蛋白质，但检测到趋势差异。与FF / HOM样品相比，OCT / LMD-TS样品的蛋白质组学特征与OCT / LMD-T样品的蛋白质组学特征更相似，表明样品处理方法的影响很大。这些结果表明，在LC-MS / MS蛋白质组学研究中，FF / HOM样品表现出与OCT / LMD样品不同的蛋白质表达谱，因此，