AuNPs/CNC Nanocomposite with A "Dual Dispersion" Effect for LDI-TOF MS Analysis of Intact Proteins in NSCLC Serum Exosomes

Detecting exosomal markers using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) is a novel approach for examining liquid biopsies of non-small cell lung cancer (NSCLC) samples. However, LDI-TOF MS is limited by low sensitivity and poor reproducibility when analyzing intact proteins directly. In this report, gold nanoparticles/cellulose nanocrystals (AuNPs/CNC) is introduced as the matrix for direct analysis of intact proteins in NSCLC serum exosomes. AuNPs/CNC with "dual dispersion" effects dispersed and stabilized AuNPs and improved ion inhibition effects caused by protein aggregation. These features increased the signal-to-noise ratio of [M+H]+ peaks by two orders of magnitude and lowered the detection limit of intact proteins to 0.01 mg mL-1. The coefficient of variation with or without AuNPs/CNC is measured as 10.2% and 32.5%, respectively. The excellent reproducibility yielded a linear relationship (y = 15.41x - 7.983, R2 = 0.989) over the protein concentration range of 0.01 to 20 mg mL-1. Finally, AuNPs/CNC-assisted LDI-TOF MS provides clinically relevant fingerprint information of exosomal proteins in NSCLC serum, and characteristic proteins S100 calcium-binding protein A10, Urokinase plasminogen activator surface receptor, Plasma protease C1 inhibitor, Tyrosine-protein kinase Fgr and Mannose-binding lectin associated serine protease 2 represented excellent predictive biomarkers of NSCLC risk. A strategy to reduce AuNP cluster and protein aggregation in the sample using AuNPs/CNC, which improved the signal sensitivity and reproducibility of LDI-TOF MS analysis for intact proteins is introduced. Through AuNPs/CNC assisted LDI-TOF MS analysis, exosome protein fingerprint can effectively distinguished between healthy individuals and NSCLC patients. Moreover, five exosomal proteins represented excellent predictive biomarkers of NSCLC risk.image