A Top-Down Proteomics Platform Coupling Serial Size Exclusion Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Mass
spectrometry (MS) based top-down proteomics provides rich
information about proteoforms arising from combinatorial amino acid
sequence variations and post-translational modifications (PTMs). Fourier
transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving
power and provides high-accuracy mass measurements, presenting a powerful
tool for top-down MS characterization of proteoforms. However, the
detection and characterization of large proteins from complex mixtures
remain challenging due to the exponential decrease in S:N with increasing molecular weight (MW) and coeluting
low-MW proteins; thus, size-based fractionation of complex protein
mixtures prior to MS analysis is necessary. Here, we directly combine
MS-compatible serial size exclusion chromatography (sSEC) fractionation
with 12 T FT-ICR MS for targeted top-down characterization of proteins
from complex mixtures extracted from human and swine heart tissue.
Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically
resolved 31 distinct proteoforms (30–50 kDa) simultaneously
in a single mass spectrum within a 100 m/z window. Notably, within a 5 m/z window, we obtained baseline isotopic resolution for 6
distinct large proteoforms (30–50 kDa). The ultrahigh resolving
power of FT-ICR MS combined with sSEC fractionation enabled targeted
top-down analysis of large proteoforms (>30 kDa) from the human
heart
proteome without extensive chromatographic separation or protein purification.
Further separation of proteoforms inside the mass spectrometer (in-MS)
allowed for isolation of individual proteoforms and targeted electron
capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR
ECD facilitated the identification and sequence characterization of
important metabolic enzymes. This platform, which facilitates deep
interrogation of proteoform primary structure, is highly tunable,
allows for adjustment of MS and MS/MS parameters in real time, and
can be utilized for a variety of complex protein mixtures.