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

Abstract

The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0–30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography–tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.

Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms

“…The large number of small proteoforms present in the sample explains also why the use of FAIMS had a major impact almost exclusively on analytes < 15 kDa. In separate studies based on the analysis of different samples such as whole cell lysates, FAIMS increased the number of proteoform identifications throughout the whole 0–30 kDa range . Therefore, we conclude that the results reported here reflect the specific compositional characteristics of the analyzed pooled serum sample.…”

“…As shown in Figure A, PEPPI is characterized by a high degree of reproducibility which is also important for the implementation of label-free quantitative TDP studies . Each protein fraction was later subjected to LC-MS 2 analysis, with or without FAIMS.…”

Targeted Quantification of Proteoforms in Complex Samples by Proteoform Reaction Monitoring

“…As a point of reference, PEPPI is often performed starting from 100 to 300 μg of protein. 25,42 Therefore, these results are not surprising. In conclusion, 75 μg of PBMCs with 1 μg of CytC is optimal for the PEPPI-PfRM assay of PBMC proteoforms.…”

“…Regarding the utility of performing targeted protein quantification at the proteoform level, it should be considered that, as reported in a recent study by Kline et al, different proteoforms derived from the same gene could show opposite expression changes (i.e., one decreasing and one increasing in abundance) when cells or tissues are subjected to specific conditions. 25 This phenomenon would likely remain unnoticed if the quantitative analysis was performed at the peptide level.…”

“…Currently, the quantitative analysis of proteoforms is performed primarily in the “discovery mode”. Following the path previously traced by bottom-up proteomics, methods for quantitative top-down proteomics in the context of high-throughput studies include both label-free − and label-based strategies (the latter comprising both metabolic and chemical labeling). − Examples of targeted quantification of proteoforms have also been described. However, these studies often involve pools of highly abundant proteoforms, are performed using the intensity of intact proteoform ions rather than fragment ions, or require lengthy sample preparation procedures (e.g., enrichment steps) to reduce background complexity and boost the signal of the targets .…”