Plasma and Serum Exosome Markers Analyzed by Matrix-Assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry Coupled with Electron Multiplier

text

May 22, 2022

Authors

Liang Shan^1,2,3; Han Gao³, Jing Zhang², Wentao Li³, Yue Su¹, Yinlong Guo²

Organizations

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

Abstract

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, R2 = 0.989; CXCL4, R2 = 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.