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 (r = 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 (n = 8, RSD = 0.7%) and 12,972 peptides (n = 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.