Tissue Mild Extraction Enriches Membrane, Cytoplasmic, and Secreted Proteomes for Biomarker Discovery

Clinical blood biomarkers provide important diagnostic
information
and are generally secreted, cytoplasmic, membrane, and other soluble
proteins. However, in the mass spectrometry-based proteomic biomarker
discovery phase, the whole tissue is often lysed directly for analysis,
and this increases protein complexity in the sample, limiting the
detection of soluble proteins as biomarkers. Here, we use a mild extraction
method that includes a low salt buffer and 1% Triton X-100 to isolate
proteins from mouse tissues followed by proteomic analyses. This mild
condition extracts membrane, cytoplasmic, and secreted proteins efficiently
while preserving nuclear proteins largely from the mouse brain and
16 other tissues. Further in-depth proteomic analysis of the mild
extract from an Alzheimer mouse brain tissue has identified more than
12,000 proteins with enrichments of soluble proteins and potential
cerebrospinal fluid markers. This mild extraction has enriched membrane,
cytoplasmic, and secreted proteins from tissue samples and should
be used as a general method for increased success in the mass spectrometry-based
proteomic discovery of circulating biomarkers.

临床血液生物标志物可提供关键诊断信息，其主要涵盖分泌蛋白、细胞质蛋白、膜蛋白及其他可溶性蛋白。然而在基于质谱的蛋白质组学生物标志物发现阶段，研究人员通常直接裂解全组织开展分析，此举会提升样品中的蛋白质复杂度，限制可溶性蛋白作为生物标志物的检测效率。本研究采用温和提取方法：以低盐缓冲液结合1% Triton X-100从小鼠组织中分离蛋白，随后进行蛋白质组学分析。该温和条件可高效提取膜蛋白、细胞质蛋白与分泌蛋白，同时在小鼠脑组织及其他16种组织中最大程度保留核蛋白。对阿尔茨海默病小鼠脑组织的温和提取产物开展深度蛋白质组学分析，共鉴定出超过12000种蛋白，其中可溶性蛋白与潜在脑脊液标志物得到显著富集。该温和提取方法可从组织样品中富集膜蛋白、细胞质蛋白与分泌蛋白，可作为通用方案提升基于质谱的循环生物标志物蛋白质组学发现研究的成功率。