Discovery proteomics by mass spectrometry comparing secreted proteins from young and old mouse lung mesenchymal stromal cells

Lung mesenchymal stromal cells (L-MSCs) were isolated from the young ( 3 months) and old (22-24 months) mice (N = 4 each) following collagenase digestion of the lungs and anchorage-dependent growth. Culture media was collected from young and old L-MSCs (10^6 cells) grown in culture for 24 hrs in serum-free condition. Culture media was then centrifuged to remove any cellular debris. Five (5) ml of supernatants were concentrated using 3kDa Molecular weight cut-off filters, and 10 micrograms of proteins were run as multiple MW 1D PAGE fractions using a standard GeLC approach with a nano-HPLC in-line with a Velos Pro Orbitrap MS. Individual data files were searched using Sequest, the results were then combined for each sample, followed by grouping, filtering, and quantifying by normalized spectral counts. Â本研究通过肺脏胶原酶消化结合贴壁依赖性生长筛选的方法，从3月龄年轻小鼠及22~24月龄老年小鼠体内分离肺间充质基质细胞（Lung mesenchymal stromal cells, L-MSCs），每组小鼠数量为4只（N=4）。将分别培养的10^6个年轻、老年L-MSCs置于无血清培养条件下培养24小时，随后收集其培养上清液。对收集到的培养上清进行离心处理，以去除残留的细胞碎片。取5mL上清液经3kDa分子量截留滤膜浓缩后，取10μg总蛋白采用标准GeLC方法进行多分子量一维聚丙烯酰胺凝胶电泳（1D PAGE）分馏，随后通过与Velos Pro Orbitrap质谱仪联机的纳流高效液相色谱（nano-HPLC）进行分析。使用Sequest软件对各原始数据文件进行检索，将每个样品的检索结果进行整合，随后通过归一化光谱计数法完成蛋白的分组、筛选与定量分析。