Enhancing Top-Down Proteomics of Brain Tissue with FAIMS

Abstract

Proteomic investigations of Alzheimer’s and Parkinson’s disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein’s “intact” state. Top-down proteomics (TDP) overcomes this limitation; however, it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, fractionation techniques are commonly used to reduce sample complexity. Here, we investigate gas-phase fractionation through high-field asymmetric waveform ion mobility spectrometry (FAIMS) within TDP. Utilizing a high complexity sample derived from Alzheimer’s disease (AD) brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS with external compensation voltage (CV) stepping at −50, −40, and −30 CV could more than double the mean number of non-redundant proteoforms, genes, and proteome sequence coverage compared to without FAIMS. We also found that FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aβ) proteoforms, including the aggregation-prone Aβ1–42 variant which is strongly linked to AD. Raw data and associated files have been deposited to the ProteomeXchange Consortium via the MassIVE data repository with data set identifier PXD023607.

Spatially resolved top-down proteomics of tissue sections based on a microfluidic nanodroplet sample preparation platform

“…While these species did not directly interfere with the analysis, their high abundance suppressed endogenous proteoform signals and reduced MS/MS time available for their characterization, outweighing the benefit of protease addition. This finding corroborates a recent TDP study (46), which mentioned some protease inhibitor cocktails branded as MS-compatible contain small proteins and should be carefully considered for TDP applications.…”

“…In these analyses, we employed FAIMS, which has been previously shown to improve proteoform coverage from bulk brain tissues. (46) The top-down workflow, illustrated in Fig. 2a, involved proteoform identification using two software tools (TopPIC (31) and TDPortal (35)); proteoform clustering to minimize redundancy using TopPICR; proteoform quantitation with ProMex; and data integration using custom R scripts.…”

Ion Mobility Spectrometry of Superheated Macromolecules at Electric Fields up to 500 Td

“…In that regard, LP-FAIMS resembles sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis where SDS in the gel simplifies protein separations by obliterating the tertiary structure . This capability may find use in top-down proteomics and tissue typing or imaging, where various AP-FAIMS systems including chips are succeeding despite poor resolution. ,,− As a filtering method with an arbitrary timescale, FAIMS is readily coupled to various MS platforms and methods including the slow FTMS and electron-transfer dissociation processes.…”

Quantitative Top-Down Proteomics by Isobaric Labeling with Thiol-Directed Tandem Mass Tags

“…Methods for increasing accuracy developed for BUP may additionally be applied for isobaric top-down experiments in the future. For example, usage of high-field asymmetric waveform ion mobility spectrometry (FAIMS) showed to increase accuracy in BUP, and its application on complex top-down samples has been recently introduced. − Furthermore, synchronous precursor selection (SPS) might also be a way to increase accuracy …”