Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA; Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA.
Anal Biochem. 2022 Oct 15;655:114846. doi: 10.1016/j.ab.2022.114846. Epub 2022 Aug 13.
Analysis of the full spectrum of secreted proteins in cell culture is complicated by leakage of intracellular proteins from damaged cells. To address this issue, we compared the abundance of individual proteins between the cell lysate and the conditioned medium, reasoning that secreted proteins should be relatively more abundant in the conditioned medium. Marked enrichment for signal-peptide-bearing proteins with increasing conditioned media to cell lysate ratio, as well loss of this signal following brefeldin A treatment, confirmed the sensitivity and specificity of this approach. The subset of proteins demonstrating increased conditioned media to cell lysate ratio in the presence of Brefeldin A identified candidates for unconventional secretion via a pathway independent of ER to Golgi trafficking.
细胞培养物中分泌蛋白的全谱分析受到受损细胞内蛋白质泄漏的影响。为了解决这个问题，我们比较了细胞裂解物和条件培养基中单个蛋白质的丰度，推断分泌蛋白在条件培养基中应该相对更丰富。随着条件培养基与细胞裂解物比值的增加，带有信号肽的蛋白质明显富集，并且在布雷菲德菌素 A 处理后丢失了这种信号，证实了这种方法的敏感性和特异性。在存在布雷菲德菌素 A 的情况下，条件培养基与细胞裂解物比值增加的蛋白质亚组鉴定出了通过非经典分泌途径的候选物，该途径独立于内质网到高尔基体的运输。
