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

Abstract

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.

Orbitrap mass spectrometry and high-field asymmetric waveform ion mobility spectrometry (FAIMS) enable the in-depth analysis of human serum proteoforms

“…While in the first FAIMS-TDP studies the CV was maintained constant throughout the whole duration of a liquid chromatography-tandem mass spectrometry (LC-MS 2 ) experiment and progressively increased across consecutive runs (a practice known as "external stepping"), [13,39] more recently Tholey and co-workers have demonstrated that an "internal stepping" strategy, whereby multiple CV values are applied in cycle within the same LC-MS 2 experiment, can be efficiently implemented, thus reducing the overall number of runs. [40,41] Our TDP analysis of two PEPPI fractions (0-15 kDa and 15-30 kDa) with and without FAIMS (with internal CV stepping) confirmed the utility of GPF via differential mobility spectrometry and demonstrated that modern Orbitrap mass spectrometry can produce the identification of more than 1000 low MW serum proteoforms from a limited number of LC-FAIMS-MS 2 runs.…”

Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry

“…Spectral simplification can counterbalance these effects. Recent studies have demonstrated that the use of field asymmetric ion mobility spectrometry (FAIMS) can lead to deeper characterization of intact proteomes in the 0–30 kDa mass range. − Three recent studies by Tholey and co-workers have first described the benefits of internal stepping of the compensatory DC voltages (compensation voltage, C.V.) applied to the central electrode of the FAIMS apparatus during a single LC–FAIMS–MS 2 analysis of proteoforms from CaCo-2 cells, , and then proven the compatibility of such data acquisition method with off-line protein fractionation via passively eluting proteins from polyacrylamide gels as intact species (PEPPI) . In the latter study, FAIMS-enabled gas-phase fractionation of intact protein ions led to the identification of more than 8500 low-molecular weight (MW) proteoforms.…”

Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling

“…SNCE schemes, however, significantly improved the identification for proteoforms with molecular weights < 20 kDa . Thus, molecular weight-based fractionation methods such as gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), , passively eluting proteins from polyacrylamide gels as intact species for MS (PEPPI-MS), , and size-exclusion chromatography (SEC) − may be considered for prefractionation so that single NCEs or more targeted SNCEs may be used for improved proteoform identification in a narrow MW range for a better balance between quantification sensitivity and identification confidence. As mentioned previously, the TMT labeling protocol was optimized for quantification of low molecular weight proteoforms; thus, sample preparation to observe larger proteins such as IgG (∼150 kDa) needs to be optimized in the future using MS-compatible detergent (e.g., Azo). ,, In this case, the optimization of NCE used for TMT-labeled larger intact proteoforms may also need to be optimized.…”