采用等压标记扩增技术对个体福尔马林固定石蜡包埋组织切片进行蛋白质组学分析。
Proteomics analysis of an individual formalin-fixed paraffin-embedded tissue section using isobaric-tag amplification.
机构信息
Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine, Seoul National University, Seoul, Republic of Korea.
Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon, Republic of Korea.
出版信息
Rapid Commun Mass Spectrom. 2023 Nov 30;37(22):e9616. doi: 10.1002/rcm.9616.
RATIONALE
The comprehensive analysis of formalin-fixed paraffin-embedded (FFPE) tissues is essential for retrospective clinical studies. However, detecting low-abundance proteins and obtaining proteome-scale data from FFPE samples pose analytical challenges in mass spectrometry-based proteomics. To overcome this challenge, our study focuses on implementing an isobaric labeling approach to improve the detection of low-abundance target proteins in FFPE tissues, thereby enhancing the qualitative and quantitative analysis.
METHODS
We employed an isobaric labeling approach utilizing synthetic peptides or proteins to enable the qualitative and quantitative measurement of target proteins in FFPE tissue samples. To achieve this, we incorporated tandem mass tag (TMT)-labeled recombinant proteins or synthetic peptides into TMT-labeled metastatic breast cancer FFPE tissues. Through this strategy, we successfully detect coexisting CD276 (B7-H3) and CD147 proteins while identifying over 6000 proteins using targeted analysis of individual FFPE tissue sections.
RESULTS
Our findings provide compelling evidence that the incorporation of isobaric labeling, along with the inclusion of TMT-labeled peptides or proteins, greatly enhances the detection of target proteins in FFPE tissue samples. By employing this approach, we were able to obtain robust qualitative measurements of CD276 and CD147 proteins, showcasing its effectiveness in identifying more than 6000 proteins in FFPE samples.
CONCLUSIONS
The integration of an isobaric labeling approach, in conjunction with synthetic peptides or proteins, presents a valuable strategy for enhancing the detection and validation of target proteins in FFPE tissue analysis. This technique holds immense potential in retrospective clinical studies, as it enables comprehensive analysis of low-abundance proteins and facilitating proteome-scale investigations in FFPE samples. By leveraging this methodology, researchers can unlock new insights into disease mechanisms and advance our understanding of complex biological processes.
背景
对福尔马林固定石蜡包埋（FFPE）组织进行综合分析对于回顾性临床研究至关重要。然而，在基于质谱的蛋白质组学中，从 FFPE 样本中检测低丰度蛋白质并获得蛋白质组规模的数据存在分析挑战。为了克服这一挑战，我们的研究集中于实施等重标记方法，以提高 FFPE 组织中低丰度靶蛋白的检测，从而增强定性和定量分析。
方法
我们采用等重标记方法，利用合成肽或蛋白质来实现 FFPE 组织样品中靶蛋白的定性和定量测量。为了实现这一目标，我们将 TMT 标记的重组蛋白或合成肽掺入 TMT 标记的转移性乳腺癌 FFPE 组织中。通过这种策略，我们成功地检测到共存的 CD276（B7-H3）和 CD147 蛋白，同时使用单个 FFPE 组织切片的靶向分析鉴定了超过 6000 种蛋白质。
结果
我们的研究结果提供了令人信服的证据，表明等重标记方法的结合，以及 TMT 标记肽或蛋白质的包含，极大地提高了 FFPE 组织样品中靶蛋白的检测。通过采用这种方法，我们能够获得 CD276 和 CD147 蛋白的稳健定性测量，展示了其在 FFPE 样品中鉴定超过 6000 种蛋白质的有效性。
结论
等重标记方法与合成肽或蛋白质的结合是一种增强 FFPE 组织分析中靶蛋白检测和验证的有价值策略。该技术在回顾性临床研究中具有巨大潜力，因为它能够全面分析低丰度蛋白质，并促进 FFPE 样本中的蛋白质组规模研究。通过利用这种方法，研究人员可以深入了解疾病机制，并推进对复杂生物过程的理解。
Zotero 插件