Native vs Denatured: An in Depth Investigation of Charge State and Isotope Distributions

Abstract

New tools and techniques have dramatically accelerated the field of structural biology over the past several decades. One potent and relatively new technique that is now being utilized by an increasing number of laboratories is the combination of socalled "native" electrospray ionization (ESI) with mass spectrometry (MS) for the characterization of proteins and their noncovalent complexes. However, native ESI-MS produces species at increasingly higher m/z with increasing molecular weight, leading to substantial differences when compared to traditional mass spectrometric approaches using denaturing ESI solutions. Herein, these differences are explored both theoretically and experimentally to understand the role that charge state and isotopic distributions have on signal-to-noise (S/N) as a function of complex molecular weight and how the reduced collisional cross sections of proteins electrosprayed under native solution conditions can lead to improved data quality in image current mass analyzers, such as Orbitrap and FT-ICR. Quantifying ion signal differences under native and denatured conditions revealed enhanced S/N and a more gradual decay in S/N with increasing mass under native conditions. Charge state and isotopic S/N models, supported by experimental results, indicate that analysis of proteins under native conditions at 100 kDa will be 17 times more sensitive than analysis under denatured conditions at the same mass. Higher masses produce even larger sensitivity gains. Furthermore, reduced cross sections under native conditions lead to lower levels of ion decay within an Orbitrap scan event over long transient acquisition times, enabling isotopic resolution of species with molecular weights well in excess of those typically resolved under denatured conditions.

Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry

“…Capable of preserving the noncovalent interactions between complexed species for further analysis, nMS extends the reach of mass spectrometry in compositional proteomics and structural biology . Native MS also simplifies complicated spectra by both populating fewer charge states and increasing m / z differences because of lower charge states relative to denaturing electrospray conditions . Two things culminate in large signal increases for nMS, especially for ions in the high kilodalton to low megadalton regime: fewer charge state channels and a lower collisional cross section .…”

Metal-Responsive Regulation of Enzyme Catalysis using Genetically Encoded Chemical Switches

“…Addition of Ni(II) to a solution of apo POP 167/517 led to a mass spectrum with a low m/z feature consistent with the presence of both one and two Ni(II) ions and a second high m/z feature consistent with the presence of a single Ni(II) ion (Figure 4c). The higher m/z of the latter feature indicates a lower charge state in which POP 167/517 residues are less accessible for ionization, as would be expected in a more closed conformation, 40 and the single metalation is consistent with coordination of Ni(II) by both BpyAla residues. Single metalation was also observed for both POP 167 and POP 517 in the presence of Ni(II), but only the low m/z feature was observed for these, consistent with metal binding with an open conformation (Supplementary Figures 8 and 9).…”

Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions

“…For each oligomer, we obtained spectra with narrow charge distributions indicative of native structure. 33 The most abundant charge states were 24+, 22+ and 21+ for Bha, Bst- Δ71 and Bst TRAP, respectively. Spectra at 12500 resolution provided baseline resolution of signals with different numbers of bound Trp.…”