Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2001-03-08
2003-11-18
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
Reexamination Certificate
active
06649907
ABSTRACT:
FIELD OF INVENTION
The present invention relates to ion sources utilizing ion-ion and ion-droplet chemical reactions to modify the charge-state distributions of ions generated by field desorption methods and in particular relates to ion sources that provide adjustable control of ion charge-state distributions produced by electrospray ionization.
BACKGROUND OF THE INVENTION
Over the last several decades mass spectrometry has advanced to the point where it has become one of the most broadly applicable analytical tools to provide fast, sensitive and selective detection of a wide variety of molecules and ions. While mass spectrometric detection provides an effective means for identifying a wide variety of molecules, its use for analyzing high molecular weight compounds is currently hindered by problems related to producing gas phase ions attributable to a given analyte species. In particular, the application of mass spectrometric analysis to determine the composition of mixtures of important biological compounds, such as oligonucleotides and oligopeptides, is severely limited by experimental difficulties related to low sample volatility and unavoidable fragmentation during vaporization and ionization processes. As a result of these limitations, the potential for quantitative analysis of samples containing biopolymers via mass spectrometry remains largely unrealized. For example, the analysis of complex mixtures of DNA molecules produced in enzymatic DNA sequencing reactions is dominated by time-consuming and labor-intensive electrophoresis techniques that may be compromised by secondary structures. The ability to selectively and sensitively detect components of complex mixtures of biological compounds via mass spectrometric methods would aid considerably in improving the accuracy, speed and reproducibility of DNA sequencing methodologies and eliminate interferences arising from secondary structure. It would also open new possibilities for the characterization of complex mixtures of proteins, carbohydrates and other polymeric species.
To be detectable via mass spectrometric methods, a compound of interest must first be produced in the form of a gas phase ion. Accordingly, it is the ion formation process which largely dictates the scope, applicability and limitations of mass spectrometry. Conventional ion preparation methods for mass spectrometric analysis have proven unsuitable for high molecular weight compounds. Vaporization by sublimation and/or thermal desorption is unfeasible for many high molecular weight compounds, including biopolymers, because these species tend to have negligibly low vapor pressures. Ionization methods based upon the desorption process, which consists of emission of ions from solid or liquid surfaces, have proven more effective in generating ions from thermally labile, nonvolatile compounds. While conventional ion desorption methods, such as plasma desorption, laser desorption, fast particle bombardment and thermospray ionization, are more applicable to nonvolatile compounds, these methods suffer from substantial problems associated with ion fragmentation and low ionization efficiencies for compounds with high molecular masses (molecular mass>2000 Dalton). To expand the applicability of mass spectrometric methods to samples containing biological compounds current research efforts have been directed toward developing new desorption and ionization methods suitable for high molecular weight species. As a result of these research efforts, two ion preparation techniques have evolved for the analysis of large molecular weight compounds; matrix assisted laser desorption and ionization-mass spectrometry (MALDI-MS) and electrospray ionization-mass spectrometry (ESI-MS).
MALDI and ESI ion preparation methods have profoundly expanded the role of mass spectrometry for the analysis of nonvolatile high molecular weight compounds including many compounds of biological interest. These ionization techniques provide high ionization efficiencies (ionization efficiency=(ions formed)/(molecules consumed)) and have been demonstrated to be applicable to biomolecules with molecular weights exceeding 100,000 Daltons. In MALDI, analyte is integrated into a crystalline organic matrix and irradiated by a short (≈10 ns) pulse of UV laser radiation at a wavelength resonant with the absorption band of the matrix molecules. Analyte molecules are entrained into a resultant gas phase plume and ionized via gas-phase proton transfer reactions occurring within the plume. While MALDI generally produces ions in singly and/or doubly charged states, significant fragmentation of analyte molecules during vaporization and ionization considerably limits the utility of MALDI as a source of gas phase ions directly attributable to a given parent compound. In addition, the sensitivity of the technique is dramatically affected by sample preparation methodology and the surface and bulk characteristics of the site irradiated by the laser. As a result, MALDI analysis is primarily used to identify the molecular masses of components of a sample and yields little information pertaining to the concentrations or molecular structures of materials analyzed.
In contrast, ESI is a field desorption ionization method that generally provides a means of generating gas phase ions with little interference from analyte fragmentation [Fenn et al., Science, 246, 64-70 (1989)]. Further, ESI provides an output consisting of a highly reproducible, continuous and homogeneous stream of analyte ions and is easily coupled to on-line liquid phase separation techniques such as high performance liquid chromatography (HPLC) and capillary electrophoresis. It is currently believed that field desorption ionization occurs by a mechanism involving strong electric fields generated at the surface of a charged substrate which extract solute analyte ions from solution into the gas phase. In ESI, a solution containing a solvent and an analyte is pumped through a capillary orifice maintained at a high electrical potential and directed at an opposing plate held near ground. The field at the capillary tip charges the surface of the emerging liquid and results in a stream of charged droplets. Subsequent evaporation of the solvent promotes a sequence of Coulombic explosions that results in droplets with a radius of curvature small enough that the electric field at their surface is large enough to desorb analyte species existing as ions in solution. Polar analyte species may also undergo desorption and ionization during electrospray by associating with cations and anions in the solution. Similar to ESI techniques, other field desorption methods have evolved that can successfully prepare ions from non-volatile, thermally liable, high molecular weight compounds. These techniques differ primarily in the physical manner in which the charged droplets are produced and include aerospray ionization, thermospray ionization and the use of pneumatic nebulization devices.
Since the ionization process proceeds via the formation of highly charged liquid droplets, ions produced by conventional field desorption methods such as ESI invariably possess a variety of multiply charged states for every analyte species discharged. Accordingly, ESI-MS spectra of mixtures are typically a complex amalgamation of peaks attributable to a large number of populated charged states for every analyte present in the sample. Therefore, ESI-MS spectra often possess too many overlapping peaks to permit effective discrimination and identification of the various components of a complex mixture. As a result of this limitation, the use of ESI-MS to analyze mixtures of biopolymers is currently severely hampered.
Recently, research efforts have been directed at expanding the utility of ESI-MS techniques for the analysis of complex mixtures of biopolymers. One method of reducing the spectral complexity of ESI-MS spectra uses computer algorithms that transform experimentally derived multiply charged spectra to “zero charge” spectra [Mann et al., Anal. Chem., 62, 1702
Ebeling Daniel D.
Scalf Mark A.
Smith Lloyd M.
Westphall Michael S.
Greenlee Winner and Sullivan P.C.
Lee John R.
Smith II Johnnie L
Wisconsin Alumni Research Foundation
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