Atsumu Hirabayashi

A sonic spray interface for the mass analysis of highly charged ions from protein solutions at high flow rates.

We have improved the sonic spray interface to enable the analysis of multiply charged ions of protein from a solution at a flow rate of 1 mL/min using a conventional liquid chromatograph/mass spectrometer. In this interface, we added a multihole plate in front of the sampling orifice of a mass spectrometer. This plate does not have a hole coaxial to the sampling orifice but has small holes around the central region of the plate. The plate reduces the density of the solvent molecules in the sprayed gas introduced into the vacuum region through the sampling orifice from the atmosphere and prevents the ions from being solvated and becoming charged droplets due to the cooling that follows adiabatic expansion of the sprayed gas. With this improvement, multiply charged ions whose charge distribution ranged from 11+ to 16+ were analyzed from a 1 μM cytochrome c solution at a high flow rate of 1 mL/min without using a splitter.

Multiply-charged Ion Formation by Sonic Spray

In sonic spray ionization technique, a solution from a fused-silica capillary is sprayed with a sonic gas flow coaxial to the capillary. The current for the ions produced and for the charged droplets is found to be dramatically enhanced when a voltage is applied to a surrounding metal piece, which is isolated from the solution by the fused-silica capillary. This can be ascribed to the induced concentration difference between positive and negative ions in the solution near the surface. Furthermore, multiply-protonated molecules produced in this way from protein solutions are analyzed with a quadrupole mass spectrometer. The ion intensity shows a strong dependence on the voltage but the diameter of the droplet produced by the spray is likely to be independent of the voltage. Thus, we conclude that the charge density of the droplet is regulated by the voltage.

Study on solvent-loading effect on inductively coupled plasma and microwave-induced plasma sources with a microliter nebulizer

Abstract To investigate the solvent-loading effect, a microliter nebulizer, called a sonic spray nebulizer (SSN), was used to introduce a sample solution at the microliter per minute level into an inductively coupled plasma (ICP) and into a nitrogen microwave-induced plasma (MIP). We compared the ICP to the MIP, and the SSN to a conventional concentric nebulizer (CCN). Our results confirm that a certain amount of water solvent is useful for analyte excitation in an Ar-ICP, since hydrogen released from the water enhances the energy transfer inside the plasma. However, we found the dominant water–solvent effect in the N2-MIP source was to consume MIP energy, rather than to promote the analyte excitation. Therefore, a means of solvent removal should be used with a N2-MIP. We also discuss other differences between the ICP and the MIP. With the SSN, several organic solvents were successfully introduced into the ICP without degrading the plasma stability.

RECENT PROGRESS IN ATMOSPHERIC PRESSURE IONIZATION MASS SPECTROMETRY

Abstract Atmospheric pressure ionization (API) is used as an interface in liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry. In API, quasi-molecular ions of biological molecules in solution are produced at atmospheric pressure. There have recently been several significant advances in spray ionization techniques, which use a solution spraying process. This article gives an overview of three spray ionization techniques: electrospray, ion spray, and sonic spray. It presents their characteristic features and describes the ion formation processes from charged droplets. Furthermore, the charged droplet formation process for each technique is described in terms of the non-uniformity in positive and negative ions in solution as well as in terms of the atomization process.

Glycopeptide identification using liquid-chromatography-compatible hot electron capture dissociation in a radio-frequency-quadrupole ion trap.

We developed a liquid chromatography (LC) compatible electron capture dissociation (ECD) mass spectrometer for glycoproteomics, with which ECD and hot ECD (HECD) experiments can be flexibly switched by quickly changing the electron energy without further tuning of the mass spectrometer. Desialylated glycopeptides were dissociated well in both ECD and HECD experiments. For sialylated glycopeptides, on the other hand, ECD with electron energy higher than 4 eV showed significantly higher sequence coverage than that with an electron energy of 0.2 eV. A nano LC system was coupled to our ECD mass spectrometer to investigate N-linked glycopeptides from lysylendopeptidase (Lys-C) digests of human transferrin. ECD spectra at multiple electron energies of 0.2, 5.0, and 9.0 eV were obtained for each targeting precursor ion in a single LC injection. Glycopeptides with a sialylated bi-, tri-, or tetra-antennary complex N-glycan were identified with high sequence coverage by HECD. Glycopeptides with tri- or tetra-antennary N-glycans have seldom been analyzed by ECD or ETD before this report. We also found that a preferential dissociation of nonreducing termini of glycans in glycopeptides by ECD and HECD.

Mass spectroscopic studies of protonation to amino-acid molecules in atmospheric pressure spray

Abstract Positive ions produced by an atmospheric pressure spray from aqueous solutions of alanine, leucine, threonine, serine, aspartic acid, and glutamic acid are detected with a double focusing mass spectrometer to study the origin of the observed ions. The relative intensities of the protonated amino-acid molecules (MH + are found to be grouped into three: (alanine and leucine)> (threonine and serine)>(aspartic and glutamic acids). This trend is ascribed to the difference in the protonation to M in the charged droplets during the spray. A qualitative explanation is proposed on the basis of the Gibbs energy of hydration.

Ion/molecule reaction and ion evaporation in atmospheric pressure spray ionization

Abstract The positive ions produced in atmospheric pressure spray ionization of ammonia, alanine and sucrose in aqueous solution were detected with a double-focusing mass spectrometer. The relative intensities of the quasi-molecular ions of ammonia, NH + 4 (H 2 O) n ( n = 0−3), were found to be proportional to the concentration of the ammonia solution and to increase with increasing distance d between the nozzle tip and the sample aperture of the mass spectrometer; this observation shows that the ammonia molecule is produced by the spray and is protonated at atmospheric pressure by a proton transfer reaction with the hydronium ion and its hydrated clusters. The observed dependences of the relative intensities of the protonated alanine molecules from alanine solution and the cationized sucrose molecules from sucrose solution on d show that some part of these quasi-molecular ions are also produced by the ion/molecule reaction in the gas phase. However, their dependences on the concentration, which are steeper than that in the ammonia case, indicate that a significant proportion of these ions are produced by ion evaporation from a droplet or liquid.

Elucidating the sequence of intact bioactive peptides by using electron capture dissociation and hot electron capture dissociation in a linear radio-frequency quadrupole ion trap.

RATIONALE Electron capture dissociation (ECD) is useful tool for sequencing of peptides and proteins with post-translational modifications. To increase the sequence coverage for peptides and proteins, it is important to develop ECD device with high fragmentation efficiency. METHODS Sequence analysis of intact undigested bioactive peptides (3000-5000 Da) was performed by use of electron capture dissociation (rf-ECD) and collision-induced dissociation (CID) in a linear radio-frequency quadrupole ion trap that was coupled to a time-of-flight mass spectrometer. We applied rf-ECD, hot rf-ECD (rf-ECD with high electron energy), and CID for intact bioactive peptide ions of various charge states and evaluated the sequence coverage of their fragment spectra. RESULTS Hot rf-ECD produced a higher number of c- and z-type fragment ions of modified peptide ions as electron energy increased in lower charged peptide ions, and sequence coverage greater than 80% was obtained compared with the CID case (40-80%). CONCLUSIONS The result indicates that intact bioactive modified peptides (Ghrelin, ANP) were correctly identified by use of hot rf-ECD.