Sequential Ion–Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer

Abstract

Obtaining extensive sequencing of an intact protein is essential in order to simultaneously determine both the nature and exact localization of chemical and genetic modifications which distinguish different proteoforms arising from the same gene. To effectively achieve such characterization, it is necessary to take advantage of the analytical potential offered by the top-down mass spectrometry approach to protein sequence analysis. However, as a protein increases in size, its gas-phase dissociation produces overlapping, low signal-to-noise fragments. The application of advanced ion dissociation techniques such as electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) can improve the sequencing results compared to slow-heating techniques such as collisional dissociation; nonetheless, even ETD-and UVPD-based approaches have thus far fallen short in their capacity to reliably enable extensive sequencing of proteoforms ≥30 kDa. To overcome this issue, we have applied proton transfer charge reduction (PTCR) to limit signal overlap in tandem mass spectra (MS 2 ) produced by ETD (alone or with supplemental ion activation, EThcD). Compared to conventional MS 2 experiments, following ETD/EThcD MS 2 with PTCR MS 3 prior to m/z analysis of deprotonated product ions in the Orbitrap mass analyzer proved beneficial for the identification of additional large protein fragments (≥10 kDa), thus improving the overall sequencing and in particular the coverage of the central portion of all four analyzed proteins spanning from 29 to 56 kDa. Specifically, PTCR-based data acquisition led to 39% sequence coverage for the 56 kDa glutamate dehydrogenase, which was further increased to 44% by combining fragments obtained via HCD followed by PTCR MS 3 .

Improving the Center Section Sequence Coverage of Large Proteins Using Stepped-Fragment Ion Protection Ultraviolet Photodissociation

“…Another promising method to enhance characterization of proteins entails the utilization of ion–ion reactions to manipulate charge states of ions. ,− Ion–ion reactions , have been performed after activation of intact proteins using proton transfer reactions (PTR) to reduce the charge states of fragment ions, thus dispersing them over a broader m / z range. ,,, This process decreases spectral congestion and improves sequence coverage of proteins, yielding an average gain of 30% coverage for bovine carbonic anhydrase II (29 kDa) in one recent study . PTR has also been used to perform gas-phase fractionations in a high-throughput manner and improve the number of identified proteoforms .…”

Improving Accuracy in Mass Spectrometry-Based Mass Determination of Intact Heterogeneous Protein Utilizing the Universal Benefits of Charge Reduction and Alternative Gas-Phase Reactions

“…For accuracy improvement, proton transfer charge reduction (PTCR), ,,, a class of gentle reactions that are not anticipated to fragment the analytes, may be used as an alternative to ETnoD/ECnoD. PTCR has been developed as a commercially available module in some models of mass spectrometers and demonstrated potential for ensemble reduction for reducing spectral complexity/overlapping of a subpopulation of fragments. , For applicability improvement, collision-induced dissociation (CID) and higher energy collisional dissociation (HCD), which are more widely available in modern instruments, may be candidates for alternative reduction reactions. CID and HCD can cleave covalent bonds in proteins and release small fragments that carry one or more charges, producing complementary residual ions in decreasing charge states.…”

Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry

“…Traditionally, the challenge of handling highly congested mass spectra was addressed by further development of mass analyzers − and signal processing methods − to increase mass resolving power and accuracy. Alternatively, charge reduction of product ions, via ion–ion reactions, reduces spectral complexity by spreading ions over a larger m / z range, thus reducing spectral congestion. − High-resolution ion mobility spectrometry (IMS) coupled with mass spectrometry (IMS-MS) offers another attractive approach in which the complexity of ion populations introduced to a mass analyzer can be reduced via gas-phase ion separations.…”