复杂的蛋白质组表征,恶化,或交联的蛋白质混合物作为配对的临床FFPE或从低血浆体积(250µL)分离的外泌体样品可能是一个挑战。在这项工作中,我们旨在研究FAIMS技术与OrbitrapExperoris480质谱仪耦合的好处,用于TMT定量蛋白质组学分析这些复杂样品,与分析细胞中的蛋白质提取物相比,冷冻组织,和从大体积血浆样品(3mL)中分离的外泌体。使用具有或不具有FAIMS和两个补偿电压(CV=-45和CV=-60)的两小时梯度LC-MS/MS进行TMT实验。在细胞的TMT实验中,冷冻组织,或使用FAIMS从大血浆体积(3mL)分离的外泌体,观察到鉴定和定量的蛋白质数量的有限增加伴随着鉴定和定量的肽数量的减少。然而,我们在此证明,与没有FAIMS的LC-MS/MS分析相比,在使用FAIMS的TMT实验中,从低血浆体积(250µL)和FFPE组织样品中分离的血浆外泌体的肽和蛋白质鉴定和定量数量有显著提高(>100%).我们的结果突出了FAIMS质谱分析的潜力,以增加深度到复杂样品的蛋白质组衍生的恶化,交联样品和/或材料稀缺的样品,如FFPE和来自低血浆体积(250微升)的血浆衍生外泌体,这可能有助于表征其蛋白质组和蛋白质形式,并鉴定可用作生物标志物的失调蛋白质。
The proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of
FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = -45 and CV = -60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with
FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with
FAIMS in comparison to the LC-MS/MS analysis without
FAIMS. Our results highlight the potential of mass spectrometry analyses with
FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.