Automated phosphopeptide enrichment from minute quantities of frozen malignant melanoma tissue

To acquire a deeper understanding of malignant melanoma (MM), it is essential to study the proteome of patient tissues. In particular, phosphoproteomics of MM has become of significant importance because of the central role that phosphorylation plays in the development of MM. Investigating clinical samples, however, is an extremely challenging task as there is usually only very limited quantities of material available to perform targeted enrichment approaches. Here, an automated phosphopeptide enrichment protocol using the AssayMap Bravo platform was applied to MM tissues and assessed for performance. The strategy proved to be highly-sensitive, less prone to variability, less laborious than existing techniques and adequate for starting quantities at the microgram level. An Fe(III)-NTA-IMAC-based enrichment workflow was applied to a dilution series of MM tissue lysates. The workflow was efficient in terms of sensitivity, reproducibility and phosphosite localization; and from only 12.5 μg of sample, more than 1,000 phosphopeptides were identified. In addition, from 60 μg of protein material the number of identified phosphoproteins from individual MM samples was comparable to previous reports that used extensive fractionation methods. Our data set included key pathways that are involved in MM progression; such as MAPK, melanocyte development and integrin signaling. Moreover, tissue-specific immunological proteins were identified, that have not been previously observed in the proteome of MM-derived cell lines. In conclusion, this workflow is suitable to study large cohorts of clinical samples that demand automatic and careful handling.为深入理解恶性黑色素瘤（malignant melanoma, MM），对患者组织的蛋白质组展开研究至关重要。尤为关键的是，由于磷酸化在恶性黑色素瘤发生发展中发挥核心作用，针对该肿瘤的磷酸化蛋白质组学研究已成为重要研究方向。然而，临床样本的研究极具挑战性，因为通常可用于开展靶向富集实验的组织材料量极为有限。本研究采用基于AssayMap Bravo平台的自动化磷酸肽富集方案处理恶性黑色素瘤组织，并对该方案的性能进行了评估。该方案灵敏度极高、变异度更低，且相较于现有技术更为简便省力，同时足以适配微克级的起始样本量。我们将基于Fe(III)-NTA-IMAC的富集流程应用于一系列梯度稀释的恶性黑色素瘤组织裂解液。该流程在灵敏度、重现性及磷酸化位点定位方面均表现优异，仅使用12.5 μg的样本即可鉴定出超过1000条磷酸肽。此外，仅需60 μg的蛋白质材料，从单个恶性黑色素瘤样本中鉴定出的磷酸化蛋白质数量，便可与此前采用深度分级分离方法的相关研究报道相媲美。本数据集涵盖了与恶性黑色素瘤进展相关的关键通路，例如丝裂原活化蛋白激酶（MAPK）、黑素细胞发育及整合素信号通路。此外，我们还鉴定出了组织特异性免疫相关蛋白，这类蛋白此前从未在恶性黑色素瘤来源细胞系的蛋白质组中被检测到。综上，该富集流程适用于需要自动化精准处理的大规模临床样本队列研究。