Grasso Giuseppe, Fragai Marco, Rizzarelli Enrico, Spoto Giuseppe, Yeo Kwon Joo
Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, Via C. Ulpiani 27, Bari, Italy.
J Mass Spectrom. 2006 Dec;41(12):1561-9. doi: 10.1002/jms.1126.
Several different procedures are available for the immobilization of proteins on solid supports, as many advantages derive from this approach, such as the possibility to develop new protein solid-state assays. Enzymes that are anchored on gold surfaces can interact with several different molecules in a tag-free environment, opening the way to surface plasmon resonance (SPR) investigations. Nevertheless, it is often important to know the identity of the affinity-retained analyte, and mass spectrometric analysis, via its unique molecular mass identification, represents a very valuable complementary method. There are many pieces of evidence to suggest that matrix metalloproteinases (MMPs) are involved in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis and cancer, but presumably also exhibiting other functions. The search for new inhibitors of MMPs has prompted research towards the development of new solid-state assays for the rapid evaluation of MMP activity. We have already reported the possibility of measuring the activity of MMP-1 anchored on solid support by coupling SPR with ESI-MS analysis. In this work, we show the in situ atmospheric pressure (AP) MALDI-MS characterization of MMPs anchored on a gold chip with known surface coverage. The study extends the MS analysis to different proteins, and sequence coverage is reported for different digestion and MS procedures.
有几种不同的方法可用于将蛋白质固定在固体支持物上，因为这种方法有许多优点，例如有可能开发新的蛋白质固态分析方法。固定在金表面的酶可以在无标签环境中与几种不同的分子相互作用，为表面等离子体共振（SPR）研究开辟了道路。然而，了解亲和保留分析物的身份通常很重要，而质谱分析通过其独特的分子量鉴定，是一种非常有价值的补充方法。有许多证据表明基质金属蛋白酶（MMPs）参与正常和病理过程，包括胚胎发生、伤口愈合、炎症、关节炎和癌症，但可能也具有其他功能。对MMPs新抑制剂的探索促使人们开展研究，以开发用于快速评估MMP活性的新固态分析方法。我们已经报道了通过将SPR与ESI-MS分析相结合来测量固定在固体支持物上的MMP-1活性的可能性。在这项工作中，我们展示了对固定在具有已知表面覆盖率的金芯片上的MMPs进行原位大气压（AP）MALDI-MS表征。该研究将质谱分析扩展到不同的蛋白质，并报告了不同消化和质谱程序的序列覆盖率。
