Jeffery Mark Brown

ETD in a Traveling Wave Ion Guide at Tuned Z-Spray Ion Source Conditions Allows for Site-Specific Hydrogen/Deuterium Exchange Measurements

The recent application of electron transfer dissociation (ETD) to measure the hydrogen exchange of proteins in solution at single-residue resolution (HX-ETD) paves the way for mass spectrometry-based analyses of biomolecular structure at an unprecedented level of detail. The approach requires that activation of polypeptide ions prior to ETD is minimal so as to prevent undesirable gas-phase randomization of the deuterium label from solution (i.e., hydrogen scrambling). Here we explore the use of ETD in a traveling wave ion guide of a quadrupole-time-of-flight (Q-TOF) mass spectrometer with a “Z-spray” type ion source, to measure the deuterium content of individual residues in peptides. We systematically identify key parameters of the Z-spray ion source that contribute to collisional activation and define conditions that allow ETD experiments to be performed in the traveling wave ion guide without gas-phase hydrogen scrambling. We show that ETD and supplemental collisional activation in a subsequent traveling wave ion guide allows for improved extraction of residue-specific deuterium contents in peptides with low charge. Our results demonstrate the feasibility, and illustrate the advantages of performing HX-ETD experiments on a high-resolution Q-TOF instrument equipped with traveling wave ion guides. Determination of parameters of the Z-spray ion source that contribute to ion heating are similarly pertinent to a growing number of MS applications that also rely on an energetically gentle transfer of ions into the gas-phase, such as the analysis of biomolecular structure by native mass spectrometry in combination with gas-phase ion-ion/ion-neutral reactions or ion mobility spectrometry.

Identifying drug metallation sites on peptides using electron transfer dissociation (ETD), collision induced dissociation (CID) and ion mobility-mass spectrometry (IM-MS)

Electron transfer dissociation (ETD) and collision induced dissociation (CID) have been used to locate the precise binding sites for platinum and ruthenium anticancer complexes on the peptide Substance P. We show that ETD combined with ion mobility-mass spectrometry significantly reduces mass spectral complexity and improves the S/N of the product-ions formed.

Continuous sample deposition from reversed‐phase liquid chromatography to tracks on a matrix‐assisted laser desorption/ionization precoated target for the analysis of protein digests

Peptide mass fingerprinting by matrix‐assisted laser desorption/ionization (MALDI)‐mass spectrometry (MS) is one of the standard high‐throughput methods for protein identification today. Traditionally this method has been based on spotting peptide mixtures onto MALDI targets. While this method works well for more abundant proteins, low‐abundance proteins mixed with high‐abundance proteins tend to go undetected due to ion suppression effects, instrumental dynamic range limitations and chemical noise interference. We present an alternative approach where liquid chromatography (LC) effluent is continuously collected as linear tracks on a MALDI target. In this manner the chromatographic separation is spatially preserved on the target, which enables generation of off‐line LC‐MS and LC‐MS/MS data by MALDI. LC‐MALDI sample collection provides improved sensitivity and dynamic range, spatial resolution of peptides along the sample track, and permits peptide mass mapping of low‐abundance proteins in mixtures containing high‐abundance proteins. In this work, standard and ribosomal protein digests are resolved and captured using LC‐MALDI sample collection and analyzed by MALDI‐TOF‐MS.

Site-Specific Analysis of Gas-Phase Hydrogen/Deuterium Exchange of Peptides and Proteins by Electron Transfer Dissociation

To interpret the wealth of information contained in the hydrogen/deuterium exchange (HDX) behavior of peptides and proteins in the gas-phase, analytical tools are needed to resolve the HDX of individual exchanging sites. Here we show that ETD can be combined with fast gas-phase HDX in ND(3) gas and used to monitor the exchange of side-chain hydrogens of individual residues in both small peptide ions and larger protein ions a few milliseconds after electrospray. By employing consecutive traveling wave ion guides in a mass spectrometer, peptide and protein ions were labeled on-the-fly (0.1-10 ms) in ND(3) gas and subsequently fragmented by ETD. Fragment ions were separated using ion mobility and mass analysis enabled the determination of the gas-phase deuterium uptake of individual side-chain sites in a range of model peptides of different size and sequence as well as two proteins; cytochrome C and ubiquitin. Gas-phase HDX-ETD experiments on ubiquitin ions ionized from both denaturing and native solution conditions suggest that residue-specific HDX of side-chain hydrogens is sensitive to secondary and tertiary structural features occurring in both near-native and unfolded gas-phase conformers present shortly after electrospray. The described approach for online gas-phase HDX and ETD paves the way for making mass spectrometry techniques based on gas-phase HDX more applicable in bioanalytical research.

Assigning structures to gas-phase peptide cations and cation-radicals. An infrared multiphoton dissociation, ion mobility, electron transfer, and computational study of a histidine peptide ion.

Infrared multiphoton dissociation (IRMPD) spectroscopy, using a free-electron laser, and ion mobility measurements, using both drift-cell and traveling-wave instruments, were used to investigate the structure of gas-phase peptide (AAHAL + 2H)(2+) ions produced by electrospray ionization. The experimental data from the IRMPD spectra and collisional cross section (Ω) measurements were consistent with the respective infrared spectra and Ω calculated for the lowest-energy peptide ion conformer obtained by extensive molecular dynamics searches and combined density functional theory and ab initio geometry optimizations and energy calculations. Traveling-wave ion mobility measurements were employed to obtain the Ω of charge-reduced peptide cation-radicals, (AAHAL + 2H)(+●), and the c(3), c(4), z(3), and z(4) fragments from electron-transfer dissociation (ETD) of (AAHAL + 2H)(2+). The experimental Ω for the ETD charge-reduced and fragment ions were consistent with the values calculated for fully optimized ion structures and indicated that the ions retained specific hydrogen bonding motifs from the precursor ion. In particular, the Ω for the doubly protonated ions and charge-reduced cation-radicals were nearly identical, indicating negligible unfolding and small secondary structure changes upon electron transfer. The experimental Ω for the (AAHAL + 2H)(+●) cation-radicals were compatible with both zwitterionic and histidine radical structures formed by electron attachment to different sites in the precursor ion, but did not allow their distinction. The best agreement with the experimental Ω was found for ion structures fully optimized with M06-2X/6-31+G(d,p) and using both projection approximation and trajectory methods to calculate the theoretical Ω values.

Extensive Charge Reduction and Dissociation of Intact Protein Complexes Following Electron Transfer on a Quadrupole-Ion Mobility-Time-of-Flight MS

AbstractNon-dissociative charge reduction, typically considered to be an unwanted side reaction in electron transfer dissociation (ETD) experiments, can be enhanced significantly in order to reduce the charge state of intact protein complexes to as low as 1+ on a commercially available Q-IM-TOF instrument. This allows for the detection of large complexes beyond 100,000 m/z, while at the same time generating top-down ETD fragments, which provide sequence information from surface-exposed parts of the folded structure. Optimization of the supplemental activation has proven to be crucial in these experiments and the charge-reduced species are most likely the product of both proton transfer (PTR) and non-dissociative electron transfer (ETnoD) reactions that occur prior to the ion mobility cell. Applications of this approach range from deconvolution of complex spectra to the manipulation of charge states of gas-phase ions. Graphical Abstractᅟ

Simple Setup for Gas-Phase H/D Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation and Ion Mobility for Analysis of Polypeptide Structure on a Liquid Chromatographic Time Scale

Gas-phase hydrogen/deuterium exchange (HDX) is a fast and sensitive, yet unharnessed analytical approach for providing information on the structural properties of biomolecules, in a complementary manner to mass analysis. Here, we describe a simple setup for ND3-mediated millisecond gas-phase HDX inside a mass spectrometer immediately after ESI (gas-phase HDX-MS) and show utility for studying the primary and higher-order structure of peptides and proteins. HDX was achieved by passing N2-gas through a container filled with aqueous deuterated ammonia reagent (ND3/D2O) and admitting the saturated gas immediately upstream or downstream of the primary skimmer cone. The approach was implemented on three commercially available mass spectrometers and required no or minor fully reversible reconfiguration of gas-inlets of the ion source. Results from gas-phase HDX-MS of peptides using the aqueous ND3/D2O as HDX reagent indicate that labeling is facilitated exclusively through gaseous ND3, yielding similar results to the infusion of purified ND3-gas, while circumventing the complications associated with the use of hazardous purified gases. Comparison of the solution-phase- and gas-phase deuterium uptake of Leu-Enkephalin and Glu-Fibrinopeptide B, confirmed that this gas-phase HDX-MS approach allows for labeling of sites (heteroatom-bound non-amide hydrogens located on side-chains, N-terminus and C-terminus) not accessed by classical solution-phase HDX-MS. The simple setup is compatible with liquid chromatography and a chip-based automated nanoESI interface, allowing for online gas-phase HDX-MS analysis of peptides and proteins separated on a liquid chromatographic time scale at increased throughput. Furthermore, online gas-phase HDX-MS could be performed in tandem with ion mobility separation or electron transfer dissociation, thus enabling multiple orthogonal analyses of the structural properties of peptides and proteins in a single automated LC-MS workflow.

Method of atmospheric pressure charge stripping for electrospray ionization mass spectrometry and its application for the analysis of large poly(ethylene glycol)s.

We introduce a new atmospheric pressure charge stripping (AP-CS) method for the electrospray ionization mass spectrometry (ESI-MS) analysis of heterogeneous mixtures, utilizing ion/ion proton transfer reactions within an experimental ion source to remove excess charge from sample ions and thereby reduce spectral congestion. The new method enables the extent of charge stripping to be easily controlled, independent of primary ionization, and there are no complications due to adduct formation. Here, we demonstrate AP-CS with a Xevo G2-S Q-TOF from Waters-Micromass using an ion source originally designed for atmospheric pressure-electron capture dissociation (AP-ECD) experiments; repurposing the AP-ECD ion source for AP-CS requires only adding a supplemental reagent (e.g., a perfluorocompound) to scavenge the electrons and generate anions for the charge-stripping reactions. Results from model peptides are first presented to demonstrate the basic method, including differences between the AP-CS and AP-ECD operating modes, and how the extent of charge stripping may be controlled. This is followed by a demonstration of AP-CS for the ESI-MS analysis of several large poly(ethylene glycol)s (PEGs), up to 40 kDa, typical of those used in biopharmaceutical development.

A parallel approach to post source decay MALDI-TOF analysis.

We present a novel enhancement to matrix-assisted laser desorption ionization (MALDI) post-source decay (PSD) analysis whereby fragment ions from multiple precursor ions are acquired into the same spectrum without employing a timed ion gate to preselect each parent ion. Fragment ions are matched to their corresponding precursor ions by comparing spectra acquired at slightly different reflectron electric fields. By measuring the difference in time-of-flight (TOF) between the two spectra for each fragment, it is possible to calculate the mass of the fragment ion and its parent. This new “parallel PSD” technique reduces analysis time and consumes less sample than conventional PSD, which requires an ion gate for serial preselection of precursor ions.

Characterization of top-down ETD in a travelling-wave ion guide.

Top-down sequencing methods are becoming increasingly relevant for protein characterization, in particular electron capture (ECD) and electron transfer dissociation (ETD) which allow for extensive backbone cleavage with minimal side reactions. The ability to obtain sequence-specific fragments while maintaining aspects of the higher-order structure, as well as the position of deuterium labels in H/D exchange, has attracted interest from scientists in the field of structural proteomics. Recently, ETD has also been combined with ion mobility on commercially available quadrupole/time-of-flight instruments, and this implementation paves the way to novel structural studies and investigation of the ETD process itself. In the current work, we investigate the use of ETD for fragmentation of standard peptides and proteins and provide a detailed description of the effect of the parameters controlling the time and efficiency of the reaction. We also highlight how the combination with ion mobility separation after electron transfer provides extended analytical benefits, such as assignment of fragments to a specific charge-reduced state of the precursor.