Parallel Analyses by Mass Spectrometry (MS) and Reverse Phase Protein Array (RPPA) Reveal Complementary Proteomic Profiles in Triple-Negative Breast Cancer (TNBC) Patient Tissues and Cell Cultures.

IF 3.4 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Proteomics Pub Date : 2024-11-16 DOI:10.1002/pmic.202400107
Nan Wang, Yiying Zhu, Lianshui Wang, Wenshuang Dai, Taobo Hu, Zhentao Song, Xia Li, Qi Zhang, Jianfei Ma, Qianghua Xia, Jin Li, Yiqiang Liu, Mengping Long, Zhiyong Ding
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Abstract

High-plex proteomic technologies have made substantial contributions to mechanism studies and biomarker discovery in complex diseases, particularly cancer. Despite technological advancements, inherent limitations in individual proteomic approaches persist, impeding the achievement of comprehensive quantitative insights into the proteome. In this study, we employed two widely used proteomic technologies, mass spectrometry (MS) and reverse phase protein array (RPPA) to analyze identical samples, aiming to systematically assess the outcomes and performance of the different technologies. Additionally, we sought to establish an integrated workflow by combining these two proteomic approaches to augment the coverage of protein targets for discovery purposes. We used 14 fresh frozen tissue samples from triple-negative breast cancer (TNBC: seven tumors versus seven adjacent non-cancerous tissues) and cell line samples to evaluate both technologies and implement this dual-proteomic strategy. Using a single-step protein denaturation and extraction protocol, protein samples were subjected to reverse-phase liquid chromatography (LC) followed by electrospray ionization (ESI)-mediated MS/MS for proteomic profiling. Concurrently, identical sample aliquots were analyzed by RPPA for profiling of over 300 proteins and phosphoproteins that are in key signaling pathways or druggable targets in cancer. Both proteomic methods demonstrated the expected ability to differentiate samples by groups, revealing distinct proteomic patterns under various experimental conditions, albeit with minimal overlap in identified targets. Mechanism-based analysis uncovered divergent biological processes identified with the two proteomic technologies, capitalizing on their complementary exploratory potential.

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质谱法 (MS) 和反相蛋白质阵列 (RPPA) 的平行分析揭示了三阴性乳腺癌 (TNBC) 患者组织和细胞培养物中互补的蛋白质组图谱。
高倍蛋白质组技术为复杂疾病(尤其是癌症)的机理研究和生物标志物发现做出了巨大贡献。尽管技术不断进步,但单个蛋白质组学方法的固有局限性依然存在,阻碍了对蛋白质组的全面定量研究。在本研究中,我们采用了两种广泛使用的蛋白质组学技术--质谱(MS)和反相蛋白质阵列(RPPA)来分析相同的样本,旨在系统地评估不同技术的结果和性能。此外,我们还试图将这两种蛋白质组学方法结合起来,建立一个综合的工作流程,以扩大蛋白质靶标的覆盖范围,从而达到发现的目的。我们使用了 14 份新鲜冷冻的三阴性乳腺癌(TNBC:7 个肿瘤和 7 个邻近的非癌组织)组织样本和细胞系样本来评估这两种技术并实施这种双重蛋白质组学策略。采用单步蛋白质变性和提取方案,对蛋白质样本进行反相液相色谱(LC),然后用电喷雾离子化(ESI)介导的 MS/MS 进行蛋白质组分析。同时,对相同的等分样品进行 RPPA 分析,以分析癌症关键信号通路或药物靶点中的 300 多种蛋白质和磷酸蛋白。这两种蛋白质组学方法都表现出了按组区分样本的预期能力,在不同的实验条件下揭示了不同的蛋白质组学模式,尽管在已确定的靶点上有极少的重叠。基于机理的分析发现了两种蛋白质组技术所确定的不同生物过程,充分利用了它们互补的探索潜力。
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来源期刊
Proteomics
Proteomics 生物-生化研究方法
CiteScore
6.30
自引率
5.90%
发文量
193
审稿时长
3 months
期刊介绍: PROTEOMICS is the premier international source for information on all aspects of applications and technologies, including software, in proteomics and other "omics". The journal includes but is not limited to proteomics, genomics, transcriptomics, metabolomics and lipidomics, and systems biology approaches. Papers describing novel applications of proteomics and integration of multi-omics data and approaches are especially welcome.
期刊最新文献
Special Issue on "Metaproteomics and meta-omics perspectives to decrypt Microbiome Functionality". In-Depth Proteome Profiling of the Hippocampus of LDLR Knockout Mice Reveals Alternation in Synaptic Signaling Pathway. Parallel Analyses by Mass Spectrometry (MS) and Reverse Phase Protein Array (RPPA) Reveal Complementary Proteomic Profiles in Triple-Negative Breast Cancer (TNBC) Patient Tissues and Cell Cultures. Review and Practical Guide for Getting Started With Single-Cell Proteomics. Omics Studies in CKD: Diagnostic Opportunities and Therapeutic Potential.
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