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

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, truncations, 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 that cannot recapitulate the original proteoform
content of samples. Clinical laboratories have been slow to adopt
top-down analysis, also due to higher sample handling requirements.
In this study, we describe a straightforward protocol for intact proteoform
sample preparation based on the depletion of albumin and immunoglobulins,
followed by simplified protein fractionation 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) to further simplify serum proteoform
mixtures. This LC-FAIMS-MS2 method led to the identification
of over 1000 serum proteoforms < 30 kDa, outperforming traditional
LC-MS2 data acquisition and more than doubling the number
of proteoforms identified in previous studies.