Abstract
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 E. coli, mouse brain, and mouse liver tissue samples, respectively. Furthermore, the successful relative label-free quantification of four spiked proteins in E. coli samples, BSA, beta-lactoglobulin, alpha-casein, and alpha-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.
