A Fast, High-Sensitivity 96-Well Plate-Based MICROFASP Method for Processing Low Microgram Proteomics Sample within 1.5 h.

A rapid, sensitive, and high-throughput sample preparation method is of paramount significance for proteomics analysis. Here, we report a fast, high-sensitivity MICROFASP method that is capable of completing sample preparation within 1.5 h, enhancing the throughput by over 13 times compared to the previous reports. Protein digestion time was significantly cut from 17 h to 20 min in a limited volume. Simultaneous reduction and alkylation occurred within 30 min. The label-free quantitation intensities of proteins from the fast and conventional MICROFASP methods were highly correlated ( = 0.91), validating the reliability of the fast-MICROFASP method. When starting with 1 μg of K562 cell lysate, the fast-MICROFASP method identified over 6 times more protein groups and 19 times more peptides than did the iST method. A 96-well plate-based version was developed to process 8 brain tissue samples from APP/PS1 transgenic mice in parallel. Averagely, with only 1 μg of protein lysate, 2826 protein groups ( = 8, RSD = 0.7%) and 12,972 peptides ( = 8, RSD = 1.5%) were identified from each sample. Amyloid-beta protein was successfully identified as a highly expressed protein, which shows its potential for detecting diagnostic markers and proteome profiling with low-microgram samples. We anticipate the high-sensitivity 96-well plate-based fast-MICROFASP method will have wide application in high-throughput and rapid preparation of large cohorts of low-microgram samples (e.g., clinical biopsy) for comprehensive proteome profiling. Data are available via ProteomeXchange with the identifier PXD053720.