Size-Based Proteome Fractionation through Polyacrylamide Gel Electrophoresis Combined with LC–FAIMS–MS for In-Depth Top-Down Proteomics

The combination of liquid chromatography (LC) and gas-phase
separation
by field-asymmetric ion mobility spectrometry (FAIMS) is a powerful
proteoform separation system for top-down proteomics. Here, we present
an in-depth top-down proteomics workflow, GeLC–FAIMS–MS,
in which a molecular-weight-based proteome fractionation approach
using SDS-polyacrylamide gel electrophoresis is performed prior to
LC–FAIMS–MS. Since individual bands and their corresponding
mass ranges require different compensating voltages (CVs), the MS
parameters for each gel band and CV were optimized to increase the
number and reliability of proteoform identifications further. We developed
an easy-to-implement and inexpensive procedure combining the earlier
established Passively Eluting Proteins from Polyacrylamide gels as
Intact species (PEPPI) protocol with an optimized Anion-Exchange disk-assisted
Sequential sample Preparation (AnExSP) method for the removal of stains
and SDS. The protocol was compared with a methanol–chloroform–water
(MCW)-based protein precipitation protocol. The results show that
the PEPPI-AnExSP procedure is better suited for the identification
of low-molecular-weight proteoforms, whereas the MCW-based protocol
showed advantages for higher-molecular-weight proteoforms. Moreover,
complementary results were observed with the two methods in terms
of hydrophobicity and isoelectric points of the identified proteoforms.
In total, 8500 proteoforms could be identified in a human proteome
standard, showing the effectiveness of the gel-based sample fractionation
approaches in combination with LC–FAIMS–MS.