Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 1. Peptides to Proteins

Abstract

Ion mobility spectrometry (IMS) coupled with hydrogen deuterium exchange (HDX)-mass spectrometry (MS) has been used to study the conformations of negatively-charged peptide and protein ions. Results are presented for ion conformers of angiotensin 1, a synthetic peptide (SP), bovine insulin, ubiquitin, and equine cytochrome c. In general, the SP ion conformers demonstrate a greater level of HDX efficiency as a greater proportion of the sites undergo HDX. Additionally, these ions exhibit the fastest rates of exchange. Comparatively, the angiotensin 1 ions exhibit a lower rate of exchange and HDX level presumably because of decreased accessibility of exchange sites by charge sites. The latter are likely confined to the peptide termini. Insulin ions show dramatically reduced HDX levels and exchange rates, which can be attributed to decreased conformational flexibility resulting from the disulfide bonds. For the larger ubiquitin and protein ions, increased HDX is observed for larger ions of higher charge state. For ubiquitin, a conformational transition from compact to more elongated species (from lower to higher charge states) is reflected by an increase in HDX levels. These results can be explained by a combination of interior site protection by compact conformers as well as decreased access by charge sites. The elongated cytochrome c ions provide the largest HDX levels where higher values correlate with charge state. These results are consistent with increased exchange site accessibility by additional charge sites. The data from these enhanced IMS-HDX experiments are described in terms of charge site location, conformer rigidity, and interior site protection.

Gas-Phase Hydrogen-Deuterium Exchange Labeling of Select Peptide Ion Conformer Types: a Per-Residue Kinetics Analysis

“…Supplementary Figure 1 shows a schematic presentation of the instrument showing home-built components (ESI source, desolvation region, and drift tube) that have been coupled to a linear ion trap (LTQ Velos; ThermoScientific, San Jose, CA, USA). The instrument configuration is similar to that described in previous reports [28,29,51]. Electrosprayed peptide ions were stored in a Bhour-glass^ion funnel device [52] (Supplementary Figure 1), where they were periodically (20 ms) pulsed into a 1-m-long drift tube filled with~2.5 Torr of 300 K He buffer gas.…”

“…HDX measurements were accomplished as described previously [9,21,28,29]. Briefly, after purifying (freeze/thaw cycles), D 2 O was added to the He buffer gas using a leak valve (Granville Phillips, Longmont, CO, USA).…”

“…Another factor that should be considered is salt bridge formation [56]. Previous IMS-HDX-MS work showed that the potential for salt bridge formation had less effect upon observed HDX levels of peptide anions compared with charge site location and conformational rigidity arising from Coulomb repulsion and disulfide bonds [29]. That said, the use of IMS-HDX with MDS where multiple structures are utilized may also reveal the influence of structural rigidity attributable to salt bridge formation.…”

“…These experiments demonstrated the ability to determine the location of deuterium uptake for selected ion conformer types and yielded a model for HDX that can be utilized in conjunction with nominal structures obtained from MDS to provide refined ion structural information. More recently, the IMS-HDX-MS approach has been used to characterize select conformations of negatively-charged peptide and protein ions [29]. This work shows an ability to decipher the roles of site accessibility (to charge sites and D 2 O) as well as conformational flexibility in the overall exchange process.…”

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

“…In the future, free electron lasers may provide atomically resolved images of solvent-free biomolecules [11], but at present other approaches have to be used. These include spectroscopic tools [12][13][14], dissociation experiments [5,6,8], gas phase H/D exchange [15,16], soft landing/EM [4,17], and computer simulations [10,[18][19][20]. The technique that is currently most widely used for characterizing biomolecular conformations in the gas phase is ion mobility spectrometry (IMS) [21][22][23][24][25][26][27].…”