V. L. Talrose Institute for Energy Problems of Chemical Physics, N. N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, Moscow, 119334, Russia.
Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia.
Biochemistry (Mosc). 2022 Nov;87(11):1342-1353. doi: 10.1134/S000629792211013X.
Protein quantitation in tissue cells or physiological fluids based on liquid chromatography/mass spectrometry is one of the key sources of information on the mechanisms of cell functioning during chemotherapeutic treatment. Information on significant changes in protein expression upon treatment can be obtained by chemical proteomics and requires analysis of the cellular proteomes, as well as development of experimental and bioinformatic methods for identification of the drug targets. Low throughput of whole proteome analysis based on liquid chromatography and tandem mass spectrometry is one of the main factors limiting the scale of these studies. The method of direct mass spectrometric identification of proteins, DirectMS1, is one of the approaches developed in recent years allowing ultrafast proteome-wide analyses employing minute-scale gradients for separation of proteolytic mixtures. Aim of this work was evaluation of both possibilities and limitations of the method for identification of drug targets at the level of whole proteome and for revealing cellular processes activated by the treatment. Particularly, the available literature data on chemical proteomics obtained earlier for a large set of onco-pharmaceuticals using multiplex quantitative proteome profiling were analyzed. The results obtained were further compared with the proteome-wide data acquired by the DirectMS1 method using ultrashort separation gradients to evaluate efficiency of the method in identifying known drug targets. Using ovarian cancer cell line A2780 as an example, a whole-proteome comparison of two cell lysis techniques was performed, including the freeze-thaw lysis commonly employed in chemical proteomics and the one based on ultrasonication for cell disruption, which is the widely accepted as a standard in proteomic studies. Also, the proteome-wide profiling was performed using ultrafast DirectMS1 method for A2780 cell line treated with lonidamine, followed by gene ontology analyses to evaluate capabilities of the method in revealing regulation of proteins in the cellular processes associated with drug treatment.
基于液相色谱/质谱法对组织细胞或生理液体中的蛋白质进行定量分析，是了解化疗治疗过程中细胞功能机制的关键信息来源之一。通过化学蛋白质组学可以获得治疗后蛋白质表达显著变化的信息，这需要分析细胞蛋白质组，并开发用于鉴定药物靶点的实验和生物信息学方法。基于液相色谱和串联质谱的全蛋白质组分析的低通量是限制这些研究规模的主要因素之一。直接质谱鉴定蛋白质的方法（DirectMS1）是近年来开发的方法之一，允许使用分钟级梯度进行分离的超快速全蛋白质组分析。本工作的目的是评估该方法在鉴定整个蛋白质组水平的药物靶点和揭示药物治疗激活的细胞过程方面的可能性和局限性。特别是，分析了先前使用多重定量蛋白质组分析方法针对一大组肿瘤药物获得的有关化学蛋白质组学的可用文献数据。将获得的结果与使用超短分离梯度通过 DirectMS1 方法获得的全蛋白质组数据进行比较，以评估该方法在鉴定已知药物靶点方面的效率。以卵巢癌细胞系 A2780 为例，对两种细胞裂解技术进行了全蛋白质组比较，包括化学蛋白质组学中常用的冻融裂解和基于超声处理的细胞破碎，后者是蛋白质组学研究中广泛接受的标准。还使用超快 DirectMS1 方法对 lonidamine 处理的 A2780 细胞系进行了全蛋白质组分析，随后进行基因本体分析，以评估该方法在揭示与药物治疗相关的细胞过程中蛋白质调控的能力。
