Effect of solvent and electrospray mass spectrometer parameters on the charge state distribution of peptides--a case study using liquid chromatography/mass spectrometry method development for beta-endorphin assay.

Beta-endorphin was used as a model peptide to study the effect of solvent and electrospray mass spectrometer parameters in the optimisation of an assay method for multiply charged compounds using liquid chromatography/mass spectrometry (LC/MS). Unlike with singly charged compounds, the charge state distribution has a significant impact in the method development of multiply charged compounds such as peptides. Using a 50% acetonitrile/water solvent mixture, we found that the ion spray voltage had no influence on the charge state distribution. However, increasing declustering potential led to deprotonation of the higher charge states of the peptide thus causing a shift to lower charge states. The mechanism leading to the deprotonation was examined. It was concluded that the deprotonation is due to endoergic proton transfer from the peptide to solvent molecules clustered to the peptide that occurs in the declustering region. The extent of deprotonation increases with increasing proton affinity of the molecules of the non-aqueous solvent component used. Thus, if desired, deprotonation can be avoided by selecting a low proton affinity solvent such as methanol. The focusing potential was also found to have a great influence on the charge state distribution observed. The results of this study enabled us to select the optimum ion to be used in single ion/reaction monitoring mode. They also provided the most favourable parameter values to be used in the method to obtain the best sensitivity for the ion of choice. The results demonstrate the importance of considering the charge state distribution in the optimisation of electrospray LC/MS methods for multiply charged compounds.

A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry.

A brief account of the mechanisms by which ions in solution are converted to ions in the gas phase is given on the basis of information available in the literature and the four companion articles on electrospray mass spectrometry (ESMS) in this issue. The following stages/phenomena are described: (a) production of the charged droplets at the ES capillary tip; (b) evolution of the charged droplets due to solvent evaporation and droplet fission caused by Coulombic repulsion of the charges on the droplets; production of the gas phase ion from very small charged droplets by the charge residue model (CRM) or the ion evaporation method (IEM); (c) dependence of the sensitivity in ESMS on the chemical nature of the analyte and its concentration as well as on the concentration of other electrolytes that are present in the solution; qualitative predictions on the sensitivity of the analyte based on the surface activity of the analyte ions; (d) relationship between ions produced in the gas phase and original ions present in the solution; and (e) globular proteins. Much of the information presented in (a)-(e) has been available for some time in the literature. However some significant advances are relatively recent. Recent results by de la Mora and co-workers, including their contribution in this Special Feature, provide very strong evidence that small ions (in distinction from macroions such as bio-macroions) are produced by IEM. On the other hand, macroions and particularly the polyprotonated globular proteins are produced by CRM. Also noteworthy is the development of an equation by Enke with which the observed relative ion signal intensities of the gas-phase ions produced can be predicted on the basis of the ion concentration in solution over a wide concentration range. The recognition that the sensitivity of organic analyte ions can be qualitatively predicted on the basis of the hydrophilicity or hydrophobicity of the part of the molecule that is not part of the charged (ionic) group and affects the surface activity of the ionic species is also noteworthy and a very useful relatively recent development.

A heated electrospray source for mass spectrometry of analytes from aqueous solutions.

An electrospray interface is described that provides high sensitivity and signal stability for mass spectrometric detection of analytes in solvents with high water content including 100% water. The electrospray capillary tip section is heated close to the boiling point of the solvent. An approximately 20°C hotter airstream, with flow coaxial to the electrospray tip and codirectional to the electrospray.. is also used. With this arrangement, the analyte signal sensitivity and stability obtained with neat water is equal to that obtained with neat methanol. The heated electrospray also affords the use of a wide range of flow rates: 1-100 µL/min.

Negative ion electrospray mass spectrometry of nucleotides: ionization from water solution with SF6 discharge suppression.

The total current and selected ion currents from the electrospray ionization (ES1) of 10(-5) M solutions of cocaine hydrochloride and deoxycytidine monophosphate (dCMP) monosodium salt in methanol and water solvents were compared in positive and negative ion modes, respectively, without and with SF6, gas as a discharge suppressant. The ESI onset voltages (Von), were the same for the positive and negative ion modes. The Von, for methanol was much lower than that for water and in agreement with the equation of D. P. H. Smith, who attributes the difference to the higher surface tension of water. The onset of electric discharge (Vdis) without SF6, occurred at lower capillary voltages for the negative relative to the positive ion mode for methanol; but Vdis is much higher than Von for methanol, and discharges do not interfere with ESI operation. For water, Von ≈ Vdis in the absence of SF6, in the negative ion mode, and ESI operation is impossible without SF6, discharge suppression. The discharge problem in the positive ion mode is less severe, but SF6, is still very useful. A dynamic range of 10 (-7)-10(-5) M was obtained by selected ion monitoring of [dCMP - H](-) at 4.5 and 20 µL/min. flows. Subpicomole detection limits for the nucleotide salt were obtained under these conditions.

Electrospray mass spectrometry of methanol and water solutions suppression of electric discharge with SF6 gas.

An equation by D. P. H. Smith predicts the capillary voltage required for the onset of electrospray (ES). For different solvents the voltage increases with the square root of the surface tension. Water requires a potential that is 1.8 times higher than that for methanol. This is verified experimentally. The higher potential required for water leads to ES in the presence of corona electric discharge. For low total ES plus corona currents, the electrosprayed analyte ion intensity is not adversely affected by the presence of discharge. At high total currents, there is a large decrease of analyte sensitivity. The sensitivity decrease is probably due to adverse space charge effect at high currents. The discharge can be suppressed by adding sulfur hexafluoride to the ambient gas. Both sensitivity and signal stability are improved. However, the sensitivity still remains lower by a factor of - 4 relative to that observed with methanol. This is attributed to lower efficiency of gas-phase ion formation from charged water, relative to methanol, droplets.

Characterization of the reaction products of deoxyguanosine with the anticancer agent BFNU and BFNU-1,1,1',1'-d4 in different buffers by high-performance liquid chromatography/atmospheric pressure ionization tandem mass spectrometry.

The products of the reaction of the anticancer agent 1,3-bis(2-fluoroethyl)-1-nitrosourea (BFNU) and BFNU-1,1,1',1'-d4 with the DNA base deoxyguanosine were characterized by applying high-performance liquid chromatography (HPLC)/tandem mass spectrometry. The total effluent from the HPLC column was introduced into the atmospheric pressure ionization (API) source of a triple-quadrupole mass spectrometer via a heated nebulizer. The gasified mixture produced from the heated nebulizer was exposed to corona discharge ionization which led to generation of gas-phase chemical ionization type of ions. The LC/API mass spectrometry produced ions and the tandem mass spectra allowed unambiguous identification, and assignment of positions of the deuterium atoms, in the products of the reaction under a variety of experimental conditions. The identification and characterization of a variety of 7-(2'-haloethyl)guanine derivatives among the reaction products provide confirmation of a proposed mechanism for the action of BFNU on DNA bases.