Istituto di Scienze e Tecnologie Chimiche (SCITEC) "Giulio Natta", Consiglio Nazionale delle Ricerche, Rome, Italy.
Department of Life Science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Fondazione Policlinico Universitario "Agostino Gemelli" Istituto di Ricovero e Cura a Carattere Scientifico, IRCCS, Rome, Italy.
J Proteomics. 2025 Jan 6;310:105339. doi: 10.1016/j.jprot.2024.105339. Epub 2024 Oct 22.
The immunomodulatory, anti-inflammatory and regenerative properties of the human amniotic mesenchymal stromal cells (hAMSCs) secretome are acknowledged but the understanding of the specific bioactive components remains incomplete. To address these limitations, the present investigation aimed to profile the proteins and peptides content of the hAMSC secretome through sample pretreatment and fractionation on 10 kDa molecular cut-off FASP (Filter Aided Sample Preparation) device and LC-MS analysis. The filter retained protein fraction underwent trypsin digestion, while the unretained was collected unchanged for intact small proteins and peptides analysis. This combined approach (C-FASP) collects in a single step two complementary fractions, advantageously saving sample volume and time of analysis. The bottom-up analysis of the C-FASP proteins fraction >10 kDa confirmed our previous findings, establishing a set of proteins consistently characterizing the hAMSC secretome. The analysis of the fraction <10 kDa, never been investigated to our knowledge, identified peptide fragments of thymosin beta 4 and beta 10, collagen alpha 1 chains I and III, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, involved in wound healing, anti-inflammatory response, tissue repair and regeneration, key biological activities of the secretome. C-FASP provided a comprehensive molecular profile of the hAMSC secretome offering new insights for enhanced therapeutic applications in regenerative medicine. SIGNIFICANCE: In this investigation we originally present the comprehensive proteomic investigation of the human amniotic mesenchymal stromal cell secretome by combining the analysis of the proteome and of the peptidome following sample pretreatment and fractionation by Filter Aided Sample Preparation (FASP) with 10 kDa molecular cut-off in coupling with LC-MS analysis. The proteome fraction retained by FASP filter was analyzed after enzymatic digestion, while the unretained fraction, below 10 kDa molecular mass, was analyzed unchanged in its intact form. This dual approach provides novel insights, previously unexplored, into the molecular components potentially responsible for the immunomodulatory and anti-inflammatory properties of the hAMSC secretome. These findings could significantly enhance the therapeutic potential of hAMSCs in regenerative medicine.
人羊膜间充质基质细胞（hAMSCs）分泌组的免疫调节、抗炎和再生特性已得到认可，但对特定生物活性成分的了解仍不完整。为了解决这些限制，本研究旨在通过 10 kDa 分子截留 FASP（Filter Aided Sample Preparation）装置对 hAMSC 分泌组的蛋白质和肽含量进行分析。截留的滤器保留了蛋白质部分，而未截留的部分则保持不变，用于分析完整的小蛋白质和肽。这种组合方法（C-FASP）在单个步骤中收集了两个互补的部分，有利地节省了样品体积和分析时间。 >10 kDa 的 C-FASP 蛋白质部分的自上而下分析证实了我们之前的发现，确定了一组一致特征化 hAMSC 分泌组的蛋白质。据我们所知，从未对 <10 kDa 的部分进行过分析，该分析确定了胸腺素β 4 和β 10、胶原α 1 链 I 和 III、α-烯醇酶和甘油醛-3-磷酸脱氢酶的肽片段，这些肽片段参与伤口愈合、抗炎反应、组织修复和再生，这是分泌组的关键生物学活性。C-FASP 提供了 hAMSC 分泌组的综合分子图谱，为再生医学中增强治疗应用提供了新的见解。 意义：在本研究中，我们通过结合 FASP（Filter Aided Sample Preparation）预处理和 10 kDa 分子截留的蛋白质组和肽组分析，首次对人羊膜间充质基质细胞分泌组进行了全面的蛋白质组学研究。FASP 滤器截留的蛋白质组在酶解后进行分析，而未截留的部分，即分子量低于 10 kDa 的部分，以完整形式进行不变分析。这种双重方法提供了新的见解，即以前未探索过的，对 hAMSC 分泌组的免疫调节和抗炎特性负责的分子成分。这些发现可以显著提高 hAMSCs 在再生医学中的治疗潜力。
