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The sampling efficiency in electrospray ionization-mass spectrometry (ESI-MS) can be improved by decreasing the liquid flow rate to the nanoflow regime, where it is possible to draw a large fraction of the ESI plume into the mass spectrometer. This mode of operation is typically more difficult than ESI-MS at higher flow rates because it requires careful optimization of a number of parameters to achieve optimal sampling efficiency. In this work, we screened the relative impact on signal intensity and spray stability of factors that included sprayer position, spray electrode protrusion, sprayer tip shape, spray angle relative to the MS inlet, nebulizer gas flow rate, ESI potential, and means for generating the electric field to initiate electrospray. Based on the screening results, we explore the possibility of providing fixed optimal values for many of the key source parameters to eliminate much of the tuning that is required for conventional nanoflow sources. This approach has potential to greatly simplify nanoflow ESI-MS, while providing optimized sensitivity, stability, and robustness, with decreased variability between analyses. [ABSTRACT FROM AUTHOR]

Sample throughput in electrospray ionization mass spectrometry (ESI-MS) is limited by the need for frequent ion path cleaning to remove accumulated debris that can lead to charging and general performance degradation. Contamination of ion optics within the vacuum system is particularly problematic as routine cleaning requires additional time for cycling the vacuum pumps. Differential mobility spectrometry (DMS) can select targeted ion species for transmission, thereby reducing the total number of charged particles entering the vacuum system. In this work, we characterize the nature of instrument contamination, describe efforts to improve mass spectrometer robustness by applying DMS prefiltering to reduce contamination of the vacuum ion optics, and demonstrate the capability of DMS to extend the interval between mass spectrometer cleaning. In addition, we introduce a new approach to effectively detect large charged particles formed during the electrospray ionization (ESI) process. [ABSTRACT FROM AUTHOR]

The properties of noncovalent complexes of the enzyme exo-1,4-β-D-glycanase ('Cex') with three aza-sugar inhibitors, deoxynojirimycin (XDNJ), isofagomine lactam (XIL), and isofagomine (XIF), have been studied with solution and gas-phase hydrogen deuterium exchange (H/Dx) and measurements of collision cross sections of gas-phase ions. In solution, complexes have lower H/Dx levels than free Cex because binding the inhibitors blocks some sites from H/Dx and reduces fluctuations of the protein. In mass spectra of complexes, abundant Cex ions are seen, which mostly are formed by dissociation of complexes in the ion sampling interface. Both complex ions and Cex ions formed from a solution containing complexes have lower cross sections than Cex ions from a solution of Cex alone. This suggests the Cex ions formed by dissociation 'remember' their solution conformations. For a given charge, ions of the complexes have greater gas-phase H/Dx levels than ions of Cex. Unlike cross sections, H/Dx levels of the complexes do not correlate with the relative gas-phase binding strengths measured by MS/MS. Cex ions from solutions with or without inhibitors, which have different cross sections, show the same H/Dx level after 15 s, indicating the ions may fold or unfold on the seconds time scale of the H/Dx experiment. Thus, cross sections show that complexes have more compact conformations than free protein ions on the time scale of ca. 1 ms. The gas-phase H/Dx measurements show that at least some complexes retain different conformations from the Cex ions on a time scale of seconds. [ABSTRACT FROM AUTHOR]

Hemoglobin (Hb) (αβ) is a tetrameric protein-protein complex. Collision cross sections, hydrogen exchange levels, and tandem mass spectrometry have been used to investigate the properties of gas-phase monomer, dimer, and tetramer ions of adult human hemoglobin (Hb A, αβ), and two variant hemoglobins: fetal hemoglobin (Hb F, αγ) and sickle hemoglobin (Hb S, αβ, E6V[β]). All three proteins give similar mass spectra. Monomers of Hb S and Hb F have similar cross sections, ca. 10% greater than those of Hb A. Cross sections of dimer ions of Hb S are 11% greater than those of Hb A and 6% greater than those of Hb F. Tetramers of Hb S are 13% larger than tetramers of Hb A or Hb F. Monomers and dimers of all three Hb have similar hydrogen-deuterium exchange (HDX) levels. Tetramers of Hb S exchange 16% more hydrogens than Hb A and Hb F. In tandem mass spectrometry, monomers of Hb S and Hb F require ca. 10% greater internal energy for heme loss than Hb A. Dimers (+11) of Hb A and Hb S dissociate to monomers with asymmetrical charge division; dimers of Hb F (+11) dissociate with nearly equal charge division. Tetramer ions dissociate to monomers and trimers, unlike solution Hb, which dissociates to dimers. The most stable dimers are from Hb S; the most stable tetramers from Hb F. The results with Hb S show that a single mutation in the β chain can change the physical properties of this gas-phase protein-protein complex. [ABSTRACT FROM AUTHOR]

Mass spectra of commercially obtained hemoglobin (Hb) show higher levels of monomer and dimer ions, heme-deficient dimer ions, and apo-monomer ions than hemoglobin freshly prepared from blood. This has previously been attributed to oxidation of commercial Hb. Further, it has been reported that that dimer ions from commercial bovine Hb have lower collision cross sections than low charge state monomer ions. To investigate these effects further, we have recorded mass spectra of fresh human Hb, commercial human and bovine Hb, fresh human Hb oxidized with HO, lyophilized fresh human Hb, fresh human Hb both lyophilized and chemically oxidized, and commercial human Hb oxidized with HO. Masses of α-monomer ions of all hemoglobins agree with the masses expected from the sequences within 3 Da or better. Mass spectra of the β chains of commercial Hb and oxidized fresh human Hb show a peak or shoulder on the high mass side, consistent with oxidation of the protein. Both commercial proteins and oxidized fresh human Hb produce heme-deficient dimers with masses 32 Da greater than expected and higher levels of monomer and dimer ions than fresh Hb. Lyophilization or oxidation of Hb both produce higher levels of monomer and dimer ions in mass spectra. Fresh human Hb, commercial human Hb, commercial bovine Hb, and oxidized commercial human Hb all give dimer ions with cross sections greater than monomer ions. Thus, neither oxidation of Hb or the difference in sequence between human and bovine Hb make substantial differences to cross sections of ions. [ABSTRACT FROM AUTHOR]

We provide modeling and experimental data describing the dominant ion-loss mechanisms for differential mobility spectrometry (DMS). Ion motion is considered from the inlet region of the mobility analyzer to the DMS exit, and losses resulting from diffusion to electrode surfaces, insufficient effective gap, ion fragmentation, and fringing field effects are considered for a commercial DMS system with 1-mm gap height. It is shown that losses due to diffusion and radial oscillations can be minimized with careful consideration of residence time, electrode spacing, gas flow rate, and waveform frequency. Fragmentation effects can be minimized by limitation of the separation field. When these parameters were optimized, fringing field effects at the DMS inlet contributed the most to signal reduction. We also describe a new DMS cell configuration that improves the gas dynamics at the mobility cell inlet. The new cell provides a gas jet that decreases the residence time for ions within the fringing field region, resulting in at least twofold increase in ion signal as determined by experimental data and simulations. [ABSTRACT FROM AUTHOR]