BIOSCOPE Research Group, LAQV-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, Universidade NOVA de Lisboa, Campus de Caparica, Caparica 2829-516, Portugal.
PROTEOMASS Scientific Society, Madan Parque, Rua dos Inventores, Caparica 2825-182, Portugal.
Anal Chem. 2020 Jul 7;92(13):9164-9171. doi: 10.1021/acs.analchem.0c01470. Epub 2020 Jun 11.
We propose a new high-throughput ultrafast method for large-scale proteomics approaches by speeding up the classic filter aided sample preparation protocol, FASP, from overnight to 2.5 h. Thirty-six samples can be treated in 2.5 h, and the method is scalable to 96-well plate-based pipelines. After a modification of the FASP-tube, the steps of protein reduction, protein alkylation, and protein digestion of complex proteomes are done in just 5.25 min, each one under the effects of an ultrasonic field (7 cycles: 30 s on and 15 s off). The new method was compared to the standard overnight digestion FASP protocol, and no statistical differences were found for more than 92.4%, 92%, and 93.3% of the proteins identified by studying the proteome of , mouse brain, and mouse liver tissue samples, respectively. Furthermore, the successful relative label-free quantification of four spiked proteins in samples, BSA, β-lactoglobulin, α-casein, and α-lactalbumin, was achieved, using either the ultrasonic-based FASP protocol or the classic overnight one. The new US-FASP method matches the analytical minimalism rules as time, cost, sample requirement, reagent consumption, energy requirements, and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all of the advantages of the filter aided sample preparation protocol are maintained.
我们提出了一种新的高通量超快方法，用于大规模蛋白质组学方法，方法是将经典的过滤辅助样品制备（FASP）方案从一晚上加速到 2.5 小时。36 个样品可以在 2.5 小时内处理，并且该方法可扩展到基于 96 孔板的流水线。对 FASP 管进行修改后，在超声场的作用下（7 个循环：30 秒开和 15 秒关），只需 5.25 分钟即可完成复杂蛋白质组的蛋白质还原、蛋白质烷基化和蛋白质消化等步骤。将新方法与标准过夜消化 FASP 方案进行比较，分别研究 、小鼠脑和小鼠肝组织样品的蛋白质组时，发现超过 92.4%、92%和 93.3%的蛋白质的鉴定没有统计学差异。此外，使用基于超声的 FASP 方案或经典的过夜方案，成功地对 样品中的 4 种添加的蛋白质（BSA、β-乳球蛋白、α-酪蛋白和α-乳白蛋白）进行了相对无标记定量。新的 US-FASP 方法符合分析极简主义规则，因为时间、成本、样品需求、试剂消耗、能源需求和废物产生都减少到最低限度，同时以稳健的方式保持高通量处理，因为保留了过滤辅助样品制备协议的所有优点。
