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 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 MScompatible 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 *

Online μSEC2-nRPLC-MS for improved sensitivity of intact protein detection of IEF separated non-human primate cerebrospinal fluid proteins

“…The choice of PolyHEA resins was motived in part by past work that showed offline serial SEC (sSEC) successfully fractionated large sarcomere proteins prior to RPLC-TDMS. 22–24 PolyHEA resin is a neutral, polar resin that can function for either hydrophilic interaction chromatography (HILIC) (mobile phase > 60 % organic) or native- or denatured-state SEC (mobile phase < 60 % organic), over broad mass ranges dependent upon pore size. 22–24, 50…”

“…Previously, SEC has been utilized in connection with studies on intact proteins for both online sample cleanup 28, 50 and offline fractionation prior to RPLC-TDMS. 24–26 Traditionally such investigations utilize analytical column diameters (e.g., ⵁ = 4.6 or 9.4 mm) at flowrates (150-500 μL/min) 21, 24 that do not readily conform to pressure limits of downstream narrow bore RP-traps/columns without use of supplemental flow splitting techniques 29 that can lead to significant sample loss. We postulated that for our workflow ( Figure 1 ), the use of SEC in the microflow (1-100 μL/min) regime (i.e., μSEC) would overcome this flow mismatch and thus permit online integration of SEC with sensitive nanoflow RPLC (nRPLC).…”

“…[18][19][20][21][22] Notably, offline SEC can fractionate complex biological samples prior to RPLC-TDMS analysis, achieving a significant increase in protein/proteoform detection over RPLC-TDMS alone by not only reducing the sample complexity but also decreasing ion suppression of HMW proteins when present in the mass spectrometer with LMW proteins. 23,24 Online SEC-MS has been achieved, [25][26][27][28][29][30][31][32][33] but its widespread use, especially high resolution separations, has been precluded by a number of technical constraints. First, there are large flow rate incompatibilities between SEC (typically 150-500 μL/min for commonly utilized column diameters of 4.6-9.4 mm) 19,22 and sensitive downstream methods, e.g., nanoflow reversed-phase liquid chromatography (nRPLC) (0.1-1.0 μL/min) coupled with electrospray ionization (ESI)-MS.…”

“…prior to RPLC-TDMS. [22][23][24] PolyHEA resin is a neutral, polar 8 resin that can function for either hydrophilic interaction chromatography (HILIC) (mobile phase > 60 % organic) or 10 native-or denatured-state SEC (mobile phase < 60 % 11 organic), over broad mass ranges dependent upon pore 12 size. [22][23][24]50 13 For the various column properties tested, calibration 14 curves fit with a third-order polynomial 44 were generated 1-3).…”

Identification and Quantification of Proteoforms by Mass Spectrometry

“…sSEC coupled to RPLC‐quadrupole‐time‐of‐flight mass spectrometry provided improved proteome coverage, with a 15‐fold improvement in the observation of proteoforms >60 kDa compared to 1D RPLC alone and enabled the observation of large proteoforms (up to 223 kDa). More recently, the Ge lab paired sSEC with fourier‐transform ion cyclotron resonance (FT‐ICR) MS analysis to enable sequence characterization of large proteoforms without RPLC separation or protein purification …”