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.