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

Jared O. Kafader, Rafael D. Melani, Luis F. Schachner, Ashley N. Ives, Steven M. Patrie, Neil L. Kelleher, Philip D. Compton

Research output: Contribution to journalArticle

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 so-called "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.

Original languageEnglish (US)
Pages (from-to)574-581
Number of pages8
JournalJournal of the American Society for Mass Spectrometry
Volume31
Issue number3
DOIs
StatePublished - Mar 4 2020

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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