Here we report a continuous flow-based ionization method, capillary Vibrating Sharp-edge Spray Ionization (cVSSI), that nebulizes liquid sample directly at the outlet of a capillary without using high-speed nebulization gas or a high electrical field. cVSSI is built upon the recently reported VSSI principle which nebulizes bulk liquid using vibrating sharp-edges. By attaching a short piece of fused silica capillary on top of the vibrating glass slide in VSSI, liquid is nebulized at the outlet of the capillary as the result of the vibration. Utilizing standard 360 μm OD/100 μm ID capillary, cVSSI works with a wide range of flow rates from 1 μL/min to 1 mL/min. The power consumption is as low as 130 mW. ESI-like MS spectra are obtained for small molecules, peptides and proteins. 5 orders of magnitude linear response for acetaminophen solution is achieved with a limit of detection (LOD) of 3 nM. cVSSI is also demonstrated to be compatible with LC-MS analysis. Two LC-MS applications are demonstrated with cVSSI: 1) separation and detection of a mixture of small molecules; 2) bottom-up proteomics using a protein digest. A mixture of 9 common metabolites was appropriately separated and detected using LC-cVSSI-MS. In the bottom-up experiment, 78 peptides were detected using LC-cVSSI-MS/MS with a protein coverage of 100% for cytochrome c, which is comparable with the coverage obtained using LC-ESI-MS. cVSSI offers a means of interfacing LC or other continuous flow-based applications to mass spectrometers with the salient features of voltage-free, flexibility, small footprint and low power consumption.
Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by the formation of amyloid fibrils of the huntingtin protein (htt). The seventeen-residue N-terminal region of htt (Nt 17 ) has been implicated in formation of early-phase oligomeric species, which may be neurotoxic. Because tertiary interactions with a downstream (C-terminal) polyproline (polyP) region of htt may disrupt oligomer formation which are precursors to fibrillar species, the effect of co-incubation of a region of htt with a 10-residue polyP peptide on oligomerization and fibrilization has been examined by atomic force microscopy (AFM). From multiple, time-course experiments, morphological changes in oligomeric species are observed for the protein/peptide mixture compared with the protein alone. Additionally, an overall decrease in fibril formation is observed for the heterogeneous mixture. To consider potential sites of interaction between the Nt 17 region and polyP, mixtures containing Nt 17 and polyP peptides have been examined by ion mobility spectrometry (IMS) and gas-phase hydrogen deuterium exchange (HDX) coupled with mass spectrometry (MS). These data combined with molecular dynamics simulations (MDS) suggest that the C-terminal region of Nt 17 may be a primary point of contact. One interpretation of the results is that polyP may possibly regulate Nt 17 by inducing a random coil region in the Cterminal portion of Nt 17 , thus, decreasing the propensity to form the reactive amphipathic α-helix. A separate interpretation is that residues important for helix-helix interactions are blocked by polyP association.
Ion mobility spectrometry-mass spectrometry (IMS-MS) in combination with gas-phase hydrogen/deuterium exchange (HDX) and collision-induced dissociation (CID) is evaluated as an analytical method for small-molecule standard and mixture characterization. Experiments show that compound ions exhibit unique HDX reactivities that can be used to distinguish different species. Additionally, it is shown that gas-phase HDX kinetics can be exploited to provide even further distinguishing capabilities by using different partial pressures of reagent gas. The relative HDX reactivity of a wide variety of molecules is discussed in light of the various molecular structures. Additionally, hydrogen accessibility scoring (HAS) and HDX kinetics modeling of candidate (in silico) ion structures is utilized to estimate the relative ion conformer populations giving rise to specific HDX behavior. These data interpretation methods are discussed with a focus on developing predictive tools for HDX behavior. Finally, an example is provided in which ion mobility information is supplemented with HDX reactivity data to aid identification efforts of compounds in a metabolite extract. Graphical Abstract ᅟ.
The dominant gas-phase conformer of [M+3H]3+ ions of the model
peptide Acetyl-PSSSSKSSSSKSSSSKSSSSK has been examined with ion mobility
spectrometry (IMS), gas-phase hydrogen deuterium exchange (HDX), and mass
spectrometry (MS) techniques. The [M+3H]3+ peptide ions are observed
predominantly as a relatively compact conformer type. Upon subjecting these ions
to electron transfer dissociation (ETD), the level of protection for each amino
acid residue in the peptide sequence is assessed. The overall per-residue
deuterium uptake is observed to be relatively more efficient for the neutral
residues than for the model peptide Acetyl-PAAAAKAAAAKAAAAKAAAAK. In comparison,
the N-terminal and C-terminal regions of the serine peptide show greater
relative protection compared with interior residues. Molecular dynamics (MD)
simulations have been used to generate candidate structures for collision cross
section and HDX reactivity matching. Hydrogen accessibility scoring (HAS) for
select structural candidates from MD simulations has been used to suggest
conformer types that could contribute to the observed HDX patterns. The results
are discussed with respect to recent studies employing extensive MD simulations
of gas-phase structure establishment of a peptide system.
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