Spray Mechanism of Contained-Electrospray Ionization.

Analytical characteristics of contained electrospray ionization (ESI) are summarized in terms of its potential to modify the analyte solution during the stages of droplet formation to provide opportunities to generate native versus denatured biomolecular gas-phase ions, without the need for bulk-phase analyte modifications. The real-time modification of the charged microdroplets occurs in a cavity that is included in the outlet of the contained-ESI ion source. Close examination of the inside of the cavity using a high-speed camera revealed the formation of discrete droplets as well as thin liquid films in the droplets wake. When operated at 20 psi N2 pressure, the droplets were observed to move at an average speed of 8 mm/s providing ∼1 s mixing time in a 10 mm cavity length. Evidence is provided for the presence of highly reactive charged droplets based on myoglobin charge state distribution, apo-myoglobin contents, and ion mobility drift time profiles under different spray conditions. Mechanistic insights for the capture of vapor-phase reagents and droplet dynamics as influenced by different operational modes are also described.

Novel manufacturing method for producing apohemoglobin and its biophysical properties.

Apohemoglobin (apoHb) is a dimeric globular protein with two vacant heme-binding pockets that can bind heme or other hydrophobic ligands. Purification of apoHb is based on partial hemoglobin (Hb) unfolding to facilitate heme extraction into an organic solvent. However, current production methods are time consuming, difficult to scale up, and use highly flammable and toxic solvents. In this study, a novel and scalable apoHb production method was developed using an acidified ethanol solution to extract the hydrophobic heme ligand into solution and tangential flow filtration to separate heme from the resultant apoprotein. Total protein and active protein yields were >95% and ~75%, respectively, with <1% residual heme in apoHb preparations and >99% purity from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Virtually no loss of apoHb activity was detected at 4°C, -80°C, and in lyophilized form during long term storage. Structurally, size exclusion chromatography (SEC) and circular dichroism indicated that apoHb was dimeric with a ~25% reduction of helical content compared to Hb. Furthermore, mass spectroscopy and reverse-phase chromatography indicated that the mass of the α and ß subunits were virtually identical to the theoretical mass of these subunits in Hb and had no detectable oxidative modifications upon heme removal from Hb. SEC confirmed that apoHb bound to haptoglobin at a similar ratio to that of native Hb. Finally, reconstituted Hb (rHb) was processed via a hemichrome removal method to isolate functional rHb for biophysical characterization in which the O2 equilibrium curve, O2 dissociation, and CO association kinetics of rHb were virtually identical to native Hb. Overall, this study describes a novel and improved method to produce apoHb, as well as presents a comprehensive biochemical analysis of apoHb and rHb.

Re-configurable, multi-mode contained-electrospray ionization for protein folding and unfolding on the millisecond time scale.

A new reconfigurable contained-electrospray (ES) ion source is described that can be operated in three unique modes (Types I, II and III) and was applied to control the charge state of proteins for subsequent online characterization by mass spectrometry. Using this device, proteins prepared in 100% water were highly charged after exposure to hydrochloric acid vapor. For myoglobin, the shift to a higher charge state occurred faster than the heme cofactor could escape the unfolding protein. This effect reflected in the detection of highly charged holo-myoglobin intermediates (+26), which suggests the modification process occurred on a millisecond time scale (Type I mode). By introducing a cavity in the contained-ES emitter (Type II mode), increased protein denaturation was observed where only apo-myoglobin ions were detected. A similar increase in the charge effect was observed for less pH sensitive proteins such as ubiquitin and cytochrome c. In a third type of experiment, acidified aqueous-based droplets containing unfolded proteins were briefly exposed to aqueous ammonium acetate/triethylammonium acetate solution mixtures to increase the droplet pH and to induce protein folding during electrospray ionization. Charge-state distributions showed protein folding occurred, including the appearance of charged reduced species that were not observed when sprayed in pure water. The three operational modes of contained-electrospray allow online modification of an analyte during charge droplet formation using both vapor and non-volatile liquid reagents, without a direct addition of the modifying reagent to the analyte solution.

Reactive Charged Droplets for Reduction of Matrix Effects in Electrospray Ionization Mass Spectrometry.

A new quantitative contained-electrospray (ES) process is described here that employs a movable ES emitter to control the reactivity of charged microdroplets by varying their exposure time with acid vapor. The method allows elimination of ion suppression effects caused by the presence of various surface active compounds that coelute with the analyte. For mixtures, contained-ESI mass spectrometric analysis produces relative ion intensities that reflect the true concentrations of analytes in solution. The mechanism for this effect has been elucidated and ascribed to the generation of fine initial droplets in the presence of a high abundance of protons; together, these two factors eliminate competition for charge and space during ion formation. Examples of analytes tested include steroids, phospholipids, phosphopeptides, and sialylated glycans. At least 1 order of magnitude improvement in detection limits, sensitivity, and accuracy of detection was observed when compared to conventional electrospray.