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

Abstract

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.

Figure 1:. Direct FT-ICR MS analysis of sSEC Fractions 9-12.

A) Top left: SDS-PAGE visualization of unfractionated soluble human heart protein extract (loading mixture, LM) and sSEC fractions 9-12. MW markers in decreasing order: 250, 150, 100, 75, 50, 37, 25, 20, 15, 10 kDa (corresponds to SDS-PAGE in Figure S1). Center: FT-ICR mass spectra acquired for LM and sSEC fractions 9-12. Segmented spectra were acquired in 100 m/z-wide quadrupole isolation windows, (window center 900, 1000, and 1100 m/z) and combined to generate a continuous overall spectrum for the LM and each sSEC fraction 9-12. Intensity for all spectra were normalized to the most intense peak in the spectrum. The protein species with the most intense signal in each mass spectrum are indicated by colored shapes (lower left-hand corner). *Ions with charge less than 3+. B) A zoom-in mass spectra (961-977 m/z) to show signal from lower-abundance species. Insets are shown for LM and fractions 9-11 to demonstrate the difference between visible protein components in each fraction.

Figure 2:. sSEC enabled detection of larger proteins after separation from the high abundance, low-MW proteins.

A) FT-ICR mass spectra for fractions 8 and 9. SDS-PAGE visualization (corresponding to Figure S1). B) A zoom-in mass spectra between 995–1012 m/z of fractions 8 and 9. Colored labels correspond to proteins with specific MWs, respectively. *oxidation products.

Figure 3:. Accurate mass measurements of metabolic enzymes by FT ICR MS following sSEC separation.

A) SDS-PAGE visualization of fractions 7 and 8 with corresponding FT-ICR mass spectra. B) A zoom-in mass spectra of 997-1001 m/z for fractions 7 and 8 showing the difference between the mass spectra of the two fractions. C) Proteins detected by intact mass from top-left to bottom-right: creatine kinase, M-type (CKM); creatine kinase, S-type (CKS); isocitrate dehydrogenase (IDH); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); malate dehydrogenase, mitochondrial (MDH); aspartate aminotransferase (AAT). External calibration was performed using DataAnalysis and theoretical fit output is generated by MASH Suite Pro using Enhanced THRASH deconvolution.

Figure 4:. ECD of Metabolic Enzymes from Soluble Heart Proteome.

A) Representative mass spectrum of 940-1040 m/z for sSEC fraction 8 of heart soluble proteome extracted from sus scrofa (swine) heart tissue. Charge states for 4 protein species are highlighted. Malate dehydrogenase, mitochondrial (MDH); creatine kinase, M-type (CKM); acetylated β -enolase (_Acβ-ENO3); glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The calculated and experimental monoisotopic masses with mass errors are indicated for each intact mass. B) Sequence maps for metabolic enzymes malate dehydrogenase, mitochondrial and creatine kinase, M-type. Each sequence table represents fragments from 1 ECD experiment. Parameters (pulse energy, pulse time, and number of transients) are indicated under each sequence table.

Scheme 1:. A schematic representation of the sSEC/FT-ICR MS top-down proteomics workflow.

1) Offline sSEC fractionation of a protein mixture with automatic fraction collection, 2) FT-ICR MS analysis of sSEC fractions, 3) In-mass spectrometer (in-MS) separation of ions for MS or MS/MS analysis, 4) Measurement of intact mass for tentative protein identification, 5) MS/MS analysis for protein characterization.