细胞外分泌的热休克蛋白90（extracellular heat shock protein 90，简称eHsp90）被认为是真核生物中的一个近期发现。过去二十年的研究提供了大量支持证据，证明eHsp90不仅在正常细胞过程如伤口愈合中起到作用，还在人类疾病和病理过程中，比如纤维化和癌症的发展中发挥作用。在2000年代早期，Eustace等人证明了eHsp90通过与并调节细胞外基质重塑酶，即基质金属蛋白酶2（matrix metalloproteinase 2，简称MMP2）的结合来促进乳腺癌细胞的侵袭。有趣的是，在哺乳动物细胞内，Hsp90是一种必需的伴侣蛋白，与数百种新合成的蛋白质（称为“客体”）相互作用，这些蛋白质需要Hsp90的协助来发挥功能。常用几种方法用于在体外和体内表征Hsp90对客体蛋白功能的作用和影响。然而，eHsp90的作用机制还没有很清晰地定义，因为迄今为止，只鉴定出了少数几种细胞外客体蛋白质。本文描述了表征这种分泌伴侣蛋白对MMP2活性的影响的方法，MMP2是最被表征的eHsp90细胞外客体。这里描述的程序可以应用和改编来表征其他细胞外客体，尤其是MMP家族的成员。 © 2023作者，Springer Science + Business Media, LLC, Springer Nature的独家许可。

Secreted, or extracellular, heat shock protein 90 (eHsp90) is considered a recent discovery in eukaryotes. Over the last two decades, studies have provided significant supporting evidence that implicates eHsp90 both in normal cellular processes such as wound healing and in the development of human pathologies and diseases including fibrosis and cancer. In the early 2000s, Eustace et al. demonstrated that eHsp90 promotes the invasion of breast cancer cells by binding to and regulating the activity of an extracellular matrix (ECM) remodeling enzyme, the matrix metalloproteinase 2 or MMP2. Interestingly, inside mammalian cells, Hsp90 is an essential chaperone that interacts with hundreds of newly synthesized proteins, known as "clients," that require Hsp90's assistance to perform their function. Several methods are routinely used to characterize the role and impact of Hsp90 on a client protein's functionality in vitro and in vivo. However, the mechanistic role of eHsp90 is less well-defined since, so far, only a handful of extracellular client proteins have been identified. Here, we describe methods to characterize the impact of the secreted chaperone on MMP2 activity, the most characterized extracellular client of eHsp90. The procedures described here can be applied and adapted to characterize other extracellular clients, particularly members of the MMP family.© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.