Ion-Ion Proton Transfer and Parallel Ion Parking for the Analysis of Mixtures of Intact Proteins on a Modified Orbitrap Mass Analyzer

Scott A Ugrin,A Michelle English,Lissa C Anderson,Dina L Bai,Jeffrey Shabanowitz,Donald F Hunt,John E P Syka

doi:10.1007/s13361-019-02290-8

Abstract

We have enabled parallel ion parking on a modified Orbitrap Elite™ as a way to control ion-ion proton transfer reactions via selective activation of a range of ions. The result is the concentration of the majority of ion current from multiple charge states of each precursor proteoform into a single charge state, maximizing signal intensity and increasing effective sensitivity compared to conventional MS1 spectra. These techniques were applied in an on-line HPLC, data-dependent MS/MS analysis of intact E. coli ribosomal proteins with HCD fragmentation. With one injection, all but two ribosomal proteins were selected for fragmentation and subsequently identified. The techniques described facilitate rapid identification of intact proteins in complex mixtures and an enhanced ability to observe proteins of low abundance.

Highlights

S tudies involving mass spectrometric analysis of intact proteins in complex mixtures are subject to many analytical challenges owing to the low abundance of many of these proteins and to the complexity of the samples analyzed
We found that such parallel ion parking (PIP) AC waveforms were not highly effective at selectively suppressing further ion-ion reactions for the relatively high charge state product ions derived from intact proteins
Attempts to increase the amplitude of the PIP waveform and thereby increase product ion kinetic excitation within the PIP m/z range resulted in collision-induced fragmentation of the charge-reduced protein product ions rather than improved suppression of further charge reduction

Summary

Introduction

S tudies involving mass spectrometric analysis of intact proteins in complex mixtures are subject to many analytical challenges owing to the low abundance of many of these proteins and to the complexity of the samples analyzed. Continued IIPT reactions will eventually generate charge-reduced products that fall outside of the available m/z range of typical mass analyzers (Supplemental Figure 1 d-e). If the reaction time is sufficiently extended, most of the product ions are “parked” at the highest product ion charge state that falls within the PIP m/z range

Methods

Results

Conclusion