Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, PR China; National Center for Organic Mass Spectrometry in Shanghai, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, PR China.
Department of Encephalopathy, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200071, PR China.
Talanta. 2022 Sep 1;247:123560. doi: 10.1016/j.talanta.2022.123560. Epub 2022 May 19.
Although matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a simple, rapid, and high-throughput assay, its microchannel plate (MCP) detector is limited by the low sensitivity and ion saturation effect when analyzing macromolecules. Herein, we introduced a strategy that combined MALDI-TOF MS with electron multiplier (EM) for the direct analysis of exosomal proteins isolated from human plasma and serum. The results demonstrated that EM yielded a higher sensitivity than MCP detector in high-mass range (m/z 5000-100000). Through the analysis of MALDI-TOF MS coupled with EM, chemokine (C-X-C motif) ligand 12 (CXCL12) ion at m/z 7960 and its degradation products at m/z 7927, 7587, and 7553 were identified as characteristic exosomal proteins in plasma. CXCL4 ion at m/z 7765 was identified as a characteristic protein in serum exosomes. Additionally, the peak intensity of CXCL12 and CXCL4 standards exhibited great linear relationship (CXCL12, R = 0.989; CXCL4, R = 0.986) with the concentrations (ranging from 0.1 to 20 μg/mL) when using EM as detector. In conjunction with ultrasonic assisted matrix coating technology (UAMCT), this assay repeatability in our lab has been excellent with coefficient of variation (CV%) of 4.6% for CXCL12 and 9.3% for CXCL4. Finally, the spectra demonstrated that the intensity of exosome related peaks was significantly enhanced in plasma and serum of patients with Parkinson's disease (PD) (m/z 7553, P < 0.01; m/z 7587, P < 0.01; m/z 7927, P < 0.001; m/z 7980, P < 0.001; m/z 7765, P < 0.01), Alzheimer's disease (AD) (m/z 7553, P < 0.001; m/z 7587, P < 0.001; m/z 7927, P < 0.001; m/z 7980, P < 0.001), and ischemic cerebrovascular disease (ICD) (m/z 7553, P < 0.05; m/z 7587, P < 0.05; m/z 7927, P < 0.01; m/z 7980, P < 0.05; m/z 7765, P < 0.05) compared to that in healthy persons. The fingerprint information of CXCL12 in plasma exosomes has better clinical relevance than serum exosome CXCL4 in MALDI-TOF MS analysis.
尽管基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）是一种简单、快速且高通量的分析方法，但当分析大分子时，其微通道板（MCP）检测器受到灵敏度低和离子饱和效应的限制。在此，我们介绍了一种将 MALDI-TOF MS 与电子倍增器（EM）相结合的策略，用于直接分析从人血浆和血清中分离的外泌体蛋白。结果表明，EM 在高质量范围（m/z 5000-100000）中比 MCP 检测器具有更高的灵敏度。通过 MALDI-TOF MS 与 EM 的分析，在 m/z 7960 处鉴定到趋化因子（C-X-C 基序）配体 12（CXCL12）离子及其在 m/z 7927、7587 和 7553 处的降解产物，作为血浆中外泌体的特征蛋白。在 m/z 7765 处鉴定到 CXCL4 离子为血清外泌体的特征蛋白。此外，当使用 EM 作为检测器时，CXCL12 和 CXCL4 标准品的峰强度与浓度（0.1-20μg/mL 范围内）呈现出极好的线性关系（CXCL12，R=0.989；CXCL4，R=0.986）。结合超声辅助基质涂覆技术（UAMCT），本实验室的该检测方法具有出色的重复性，对于 CXCL12 和 CXCL4 的变异系数（CV%）分别为 4.6%和 9.3%。最后，图谱表明，帕金森病（PD）（m/z 7553，P<0.01；m/z 7587，P<0.01；m/z 7927，P<0.001；m/z 7980，P<0.001；m/z 7765，P<0.01）、阿尔茨海默病（AD）（m/z 7553，P<0.001；m/z 7587，P<0.001；m/z 7927，P<0.001；m/z 7980，P<0.001）和缺血性脑血管病（ICD）（m/z 7553，P<0.05；m/z 7587，P<0.05；m/z 7927，P<0.01；m/z 7980，P<0.05；m/z 7765，P<0.05）患者血浆中外泌体相关峰的强度明显高于健康人。与 MALDI-TOF MS 分析中的血清外泌体 CXCL4 相比，血浆外泌体 CXCL12 的指纹信息具有更好的临床相关性。
