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

20 February 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncation, single amino acid substitutions and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches which cannot recapitulate the original proteoform content of samples. Lengthy sample preparation and the need for extensive prefractionation to mitigate proteoform dynamic range issues are likely factors preventing clinical laboratories from routinely performing top-down experiments. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on depletion of albumin and immunoglobulins followed by simplified fractionation of remaining serum proteins via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS), which served as an additional separation dimension to further simplify serum proteoforms mixtures. This LC-FAIMS-MS2 method led to the identification of over 1,000 serum proteoforms <30 kDa using a reduced number of experiments, more than doubling the number of proteoforms identified in previous studies.


mass spectrometry

Supplementary materials

Supporting Information: 1. Table S1 2. Figures S1-S3


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