On the Scalability and Requirements of Whole Protein Mass Spectrometry

Abstract

Top-down proteomics has improved over the last decade despite the significant challenges presented by the analysis of large protein ions. Here, the detection of these high mass species by electrospray-based mass spectrometry (MS) is examined from a theoretical perspective to understand the mass-dependent increases in the number of charge states, isotopic peaks, and interfering species present in typical protein mass spectra. Integrating these effects into a quantitative model captures the reduced ability to detect species over 25 kDa with the speed and sensitivity characteristic of proteomics based on <3 kDa peptide ions. The model quantifies the challenge that top-down proteomics faces with respect to current MS instrumentation and projects that depletion of 13C and 15N isotopes can improve detection at high mass by only <2-fold at 100 kDa whereas the effect is up to 5-fold at 10 kDa. Further, we find that supercharging electrosprayed proteins to the point of producing <5 charge states at high mass would improve detection by more than 20 fold.

Top Down proteomics: Facts and perspectives

“…Extremely high resolution may be required to distinguish disulfide bridges (Δm = 2 Da), deamidation (Δm = 1 Da), trimethylation versus acetylation (Δm = 39 mDa), and phosphorylation versus sulfation (Δm = 10 mDa) [70]. Sensitivity is also vital, as high molecular weight species such as proteins will have broad isotopic distributions, distributing the signal from a single protein across many peaks [71]. Additionally, in electrospray ionization (ESI) a population of protein ions will display a distribution of charge states.…”

Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

“…This relationship becomes more consequential when analyzing intact proteins, as larger proteins tend to be more highly charged in standard electrospray ionization. This connection between charge state distribution and protein mass has been empirically modeled by Kelleher and co-workers [27]; according to their work, the theoretical charge state distribution of a protein as function of its molecular weight (MW) can be estimated as: $$C{S}_n=e^{false[-{false(n-4.12\times {10}^{-4}false(MWfalse)-0.297false)}^2/2{false(1.59\times {10}^{-4}false(MWfalse)-0.153false)}^{2}false]}$$ $$\text{for}0<n<false\lfloor 8.64\times {10}^{-4}false(MWfalse)+1false\rfloor$$…”

“…To compound S/N challenges further, larger proteins generate larger fragments, which have broader isotope distributions. Depreciation in peak abundance due to the presence of naturally occurring isotopes can be expressed by the relationship: $$S/N\propto 1/\sqrt{MW}$$ which ultimately requires a greater number of ions for larger fragments to raise usable signal above the noise band (Figure 1d) [27, 56]. …”

“…The tradeoff for the increase in S/N , however, is a significant increase in acquisition times required for generation of high quality spectra, accordingly limiting the sampling depth achievable in a given experiment. We conclude that increasing the number of precursor ion charges prior to initiation of the dissociation event is a direct way to improve S/N without spectral averaging [27, 48, 49]. Herein we describe modifications to ion processing and storage that permit increased precursor ion populations for ETD experiments – we call this method high capacity ETD (also called ETD High Dynamic range, or ETD HD).…”

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics

“…The limitations in denaturing mode are related to instrumentation, reduction of signal because of multiple charge states, chemical noise, incomplete desolvation and the presence of multiple PTMs like glycosylation that can frustrate the detection of such signals (65). The use of a native fractionation technique coupled to native MS/MS mitigated many of these problems and proved to be an efficient method to analyze the largest proteins present in the venom.…”