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 ultra-high resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, 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 co-eluting 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 the human and swine heart proteome. Benefiting from the ultra-high 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 ultra-high 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 of the mass spectrometer (in-MS) allowed for isolation of individual proteoforms for targeted electron capture dissociation (ECD) for high sequence coverage. sSEC/FT-ICR ECD facilitated 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.