Profiling of Native and Protease_Generated Protein Amino Termini from Formalin-Fixed, Paraffin-Embedded Tissues

Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N-termini from FFPE specimens. We demonstrate a comparable N-terminomes of cryopreserved and formalin fixed tissue, thereby showing that formalin fixation / paraffin embedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identified a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies.

蛋白水解失调是诸多疾病的标志性特征之一。近年来，越来越多的研究开始聚焦蛋白末端，以期在临床研究中揭示蛋白酶的生理与病理功能。然而，冷冻保存组织样本的可获取性往往受限。而福尔马林固定石蜡包埋（formalin-fixed, paraffin-embedded, FFPE）组织则为临床研究提供了极为宝贵的研究资源。与病理相关的组织通常以FFPE形式储存，是归档人类样本中最丰富的资源类型。本研究建立了一套稳定的实验流程，用于探究FFPE样本中天然存在的以及蛋白酶酶解产生的蛋白N末端。本研究证实冷冻保存组织与福尔马林固定组织的N末端组学（N-terminome）特征具有可比性，表明福尔马林固定与石蜡包埋过程不会对蛋白造成酶解损伤。因此，FFPE样本完全适用于蛋白N末端分析。此外，我们在针对组织蛋白酶L（cathepsin L）缺陷型与野生型小鼠的FFPE肝脏样本的定量N末端组学研究中，证实了FFPE降解组学（FFPE-degradomics）的可行性。结合机器学习方法与此前已明确的组织蛋白酶L底物特异性，我们成功鉴定出多个潜在的组织蛋白酶L切割位点。本研究证实，FFPE样本可作为降解组学研究中冷冻保存组织的优质替代资源。