BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow

The
GeLC–MS workflow, which combines low-cost, easy-to-use
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE)
with liquid chromatography–mass spectrometry (LC–MS),
is very popular in current bottom-up proteomics. However, GeLC–MS
requires that PAGE-separated proteins undergo overnight enzymatic
digestion in a gel, resulting in more than 20 h of sample preparation
for LC–MS. In this study, we overcame the limitations of GeLC–MS
by developing a rapid digestion workflow for PAGE separation of proteins
using N,N′-bis­(acryloyl)­cystamine
(BAC) cross-linked gels that can be solubilized by reductive treatment.
Making use of an established workflow called BAC-DROP (BAC-gel dissolution
to digest PAGE-resolved objective proteins), crude proteome samples
were fractionated based on molecular weight by BAC cross-linked PAGE.
After fractionation, the gel fragments were reductively dissolved
in under 5 min, and in-solution trypsin digestion of the protein released
from the gel was completed in less than 1 h at 70 °C, equivalent
to a 90–95% reduction in time compared to conventional in-gel
trypsin digestion. The introduction of the BAC-DROP workflow to the
MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed
for serum sample preparation to be completed in as little as 5 h,
demonstrating successful marker quantification from a 0.5 μL
sample of human serum.