A fast, high-sensitivity 96-well plate based MICROFASP method for processing low microgram proteomics sample within 1.5 hours

We report a fast, high-sensitivity MICROFASP method that capable of completing sample preparation in less than 1.5 h which improved the throughput over 13 times compared to the previous reports. In this method, protein digestion time was dramatically shortened from 17 h to 20 min in the limited reaction volume. To further decrease the sample preparation time, we used tris(2-carboxyethyl)phosphine and Iodoacetamide (IAA) for the reduction of disulfide bonds and the alkylation of the resulted free thiol groups simultaneously instead of using dithiothreitol and IAA which has to be carried out the reactions in two separate steps. Label free quantitation (LFQ) intensities of the proteins identified from the fast MICROFASP method and the conventional MICROFASP method were highly correlated (the pearson correlation coefficient r=0.907) which confirmed the reliability of the fast MICROFASP method. We also developed 96-well plate based fast-MICROFASP method to process 8 brain tissue samples from APP/PS1 transgenic mice in parallel. Amyloid-beta protein, a pathological protein of Alzheimer’s disease, was successfully identified as the high expressed protein in the brain of APP/PS1 transgenic mice, suggesting the diagnositic potential of our fast-MICROFASP method. We anticipate the fast, high-sensitivity 96-well plate based MICROFASP method has wide application in high-throughput and rapid preparation of large cohorts of low microgram samples (e.g., clinical biopsy) for comprehensive proteome profiling.