Accumulating evidence suggests that solution-phase conformations of small globular proteins and large molecular protein assemblies can be preserved for milliseconds after electrospray ionization. Thus, the study of proteins in the gas-phase on this time-scale is highly desirable. Here we demonstrate that a travelling wave ion guide (TWIG) of a Synapt mass spectrometer offers a highly suitable environment for rapid and efficient gas-phase hydrogen/deuterium exchange (HDX). Gaseous ND 3 was introduced into either the source TWIG or the TWIG located just after the ion mobility cell, such that ions underwent HDX as they passed through the ND 3 on the way to the timeof-flight analyzer. The extent of deuterium labeling could be controlled by varying the quantity of ND 3 or the speed of the travelling wave. The gas-phase HDX of model peptides corresponded to labeling of primarily fast exchanging sites due to the short labeling times (ranging from 0.1 to 10 ms). In addition to peptides, gas-phase HDX of ubiquitin, cytochrome c, lysozyme and apomyoglobin were examined. We conclude that HDX of protein ions in a TWIG is highly sensitive to protein conformation, enables the detection of conformers present on sub-milliseconds timescales and can readily be combined with ion mobility spectrometry.

Top-down approaches for the characterization of intact proteins and macromolecular complexes are becoming increasingly popular, since they potentially simplify and speed up the assignment process. Here we demonstrate how, on a commercially available Q-TWIMS-TOF instrument, we performed top-down ETD of the native form of tetrameric alcohol dehydrogenase. We achieved good sequence coverage throughout the first 81 N-terminal amino acids of ADH, with the exception of a loop located on the inside of the protein. This is in agreement with the exposed parts of the natively folded protein according to the crystal structure. Choosing the right precursor charge state and applying supplemental activation were found to be key to obtaining a high ETD fragmentation efficiency. Finally, we briefly discuss opportunities to further increase the performance of ETD based on our results.

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.

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.

An ion mobility mass spectrometer has been modified to allow optical interrogation of ions with different mass-to-charge (m/z) ratios and/or mobilities (K). An ion gating and trapping procedure has been developed which allows us to store ions for several seconds enabling UV photodissociation (UVPD).

Abstract. Non-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.

Matrix-assisted laser desorptiodionization combined with collision-induced dissociation (CID) has been applied to the structural determination of synthetic polymers. Post-source decaylCID experiments on a time-of-flight (TOF) instrument have been compared with CID data from a hybrid sector-TOF mass spectrometer. Fragmentation spectra of polymers, with molecular weights of up to 4500Da, have been shown to aid structural and end-group determination. The polymers studied were poly(methy1 methacrylate), poIy(ethy1ene glycol) and poly(ethy1ene terephthalate).Tandem mass spectrometry (MSMS) has made a big impact in the biological area,'.' especially when used in conjunction with soft ionization techniques such as liquid secondary ion mass spectrometry (LSlMS)3.4 and, more recently, electrospray ionization (ESI).5*6 Many synthetic polymers, however, are not amenable to ionization by these techniques or else the ion currents obtained are too low, making the generation of fragment-ion spectra difficult.Collision-induced dissociation (CID) spectra, generally from species with molecular weights of below lOOODa, have been shown for ions of polymers generated by LSIMS"I3, field de~orption'~. l5 and ESI.'" l7 Structural12* l5 and end-group information13 has been observed in CID spectra of polymers with molecular weights of less than 2500 Da.The matrix-assisted laser desorptiodionization (MALDI) technique has recently been shown to be a useful tool for the direct analysis of synthetic polymers,'&-20 but has typically been used with time-of-flight (TOF) instruments which did not have the capability for MSMS experiments. This paper describes the first application of MALDI-CID to the analysis of synthetic polymers. Spectra are shown for oligomers of poly(methylmethacry1ate) (PMMA) poly-(ethyleneterephthalate) (PET) and poly(ethy1ene glycol (PEG) with molecular weights of 500-4500 Da. EXPERIMENTALMass spectrometry MALDI-CID experiments were performed using an AutoSpec 5000 orthogonal acceleration (oa)-TOF (Micromass, Manchester, UK) tandem mass spectrometer equipped with a MALDI source. This hybrid, sector-oa-TOF instrument has been described in more detail elsewhere.2' The nitrogen laser (A=337 nm) was operated at a pulse rate of 10 Hz in the MALDI source. The precursor ions, accelerated by a voltage of 8 kV, were selected by MS-I (a double-focusing (EBE) mass spectrometer). These ions were decelerated to an energy of 800 eV and focused into the collision cell. The Author for correspondence. precursor ion beam intensity was attenuated by approximately 70% using xenon as the collision gas. Ions leaving the collision cell were directed into the oa-TOF analyser (MS-2) and, because of the pulsed nature of the MALDI technique, the voltage pulse applied to the oa-TOF was automatically timed to coincide with the time at which the packet of precursor and product ions was passing through the orthogonal acceleration chamber. The product ions were detected by the microchannel plate detector which has a total length of 150 mm. The full produc...

The initial stages of protein unfolding may reflect the stability of the entire fold and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work, we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation; experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins, the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M + 7H], cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions whilst cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles. Graphical Abstract.