Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2001-03-23
2004-04-27
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C250S282000
Reexamination Certificate
active
06727497
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to electrospray ionization mass spectrometry, and more particularly to a method of charge reduction whereby ions produced by electrospray are amenable to partial neutralization and subsequent detection by an orthogonal time-of-flight mass spectrometer to yield high resolution mixture spectra.
2. Description of Related Art
The structure of deoxyribonucleic acid (DNA) consists of two parallel strands connected by hydrogen bonding. Double stranded DNA molecules assume a double helix structure with varying geometric characteristics. Under certain salt or temperature conditions, denaturation can occur and the two DNA strands become separated.
The order of nucleotides along a single strand corresponds to the sequence of DNA. Each set of three contiguous bases (a codon) encodes a particular amino acid used in protein synthesis. Successive codons are organized into a gene to encode a particular protein. DNA is thus present in living cells as the fundamental genetic information carrier.
The human genome is the complete set of human DNA present in every cell (apart from reproductive and red blood cells). It is believed that total human DNA comprises 3 billion base pairs encoding about 100,000 genes. Sequencing the entire genome is desirable because knowledge of gene sequencing should increase the understanding of gene regulation and function and allow precise diagnostics and treatment of genetic diseases.
Using current sequencing technologies, about 14,000 base pairs can be acquired in 14 hours in an electrophoresis gel. The ultimate goal of 3 billion base pairs therefore poses a technological challenge and presents a need for high performance sequencing instruments. To this end, mass spectrometry can be used as a sequencing technique.
An important field emerging from genomics is proteomics. Proteomics concerns the study of all the proteins encoded for by genes. Like genomics, proteomics involves extremely complex mixtures of large biopolymers (proteins in this case) that need to be separated and identified. Current technologies mainly make use of 2-D electrophoresis gels, which separate proteins based on both size and the isolelectric point of the proteins. These gels are labor intensive to prepare and time-consuming to run and analyze. Mass spectrometry offers a high-speed, high-sensitivity, low-labor alternative to separate, sequence, and identify complex mixtures of proteins.
Mass spectrometry allows the acquisition of molecular weights (measured in daltons) for every mass to charge (m/z) peak acquired, whereby the m/z ratio is an intrinsic and condition-independent property of an ion. By eliminating the preparation of gels required with electrophoretic mobility analysis, mass spectrometry has the potential for requiring only milliseconds per analysis. By its nature, it is an intrinsically fast and accurate means for accurately assessing molecular weights.
Mass spectrometry requires that the analyte of interest be produced in the form of a gas phase ion, within the vacuum of a mass spectrometer for analysis. While achieving this is straightforward for small molecules using classical techniques (such as sublimation or thermal desorption) used in conjunction with an ionization method (such as electron impact), it is much less straight-forward for large biopolymers with essentially nonexistent vapor pressures. For this reason, the field of large-molecule mass spectrometry was extremely limited for many years. This situation changed dramatically with the discovery of two important new techniques for producing ions of large biomolecules (macromolecules), namely Matrix Assisted Laser Desorption-lonization (MALDI) and Electrospray Ionization (ESI), whereby rapidly determining the mass of large molecules became feasible.
In MALDI mass spectrometry, a few hundred femtomoles of analyte are mixed on a probe tip with a small, organic, ultra-violet (UV) absorbing compound, the matrix. The analyte-matrix is dried to produce a heterogenous crystalline dispersion, and then irradiated with a brief (i.e., 10 ns) pulse of UV laser radiation in order to volatilize the sample and produce gas phase ions of the analyte amenable to mass spectrometric analysis. Because the UV pulse is at a wavelength that is absorbed by the matrix and not the analyte, the matrix is vaporized, and analyte molecules become entrained in the resultant gas phase plume where they are ionized in gas phase proton transfer reactions. However, analyte fragmentation and poorly understood matrix effects occur during the MALDI process, thereby reducing molecular ion intensity and complicating the analysis and interpretation of the mass spectra. As a result, the mass range of this technique is limited; it frequently does not allow sequencing fragments longer then 35-100 base pairs in length.
Electrospray ionization mass spectrometry (ESI-MS), on the other hand, allows analysis of DNA with reduced fragmentation. ESI-MS is characterized by a gentle analyte desorption process that can leave noncovalent bonds intact. This soft ionization allows analysis of intact DNA molecular ions. However, ESI-MS typically produces multiply charged ions, and as the number of possible charge states increases with the size of the analyte, this technique yields complex spectra for large molecules. For example, while ESI analysis of simple molecules may be accomplished using computer algorithms that transform the multiply charged mass spectra to “zero-charge” spectra, permitting easy visual interpretation thereof, as spectral complexity and chemical noise levels increase, these algorithms produce artificial peaks and miss analyte peaks with low signal intensity. Furthermore, each analyte yields a specific peak distribution and mixture spectra are therefore characterized by complex overlapping distributions for which the resultant spectra cannot be resolved without expensive high resolution mass spectrometers. This multiple charging and peak multiplicity in ESI-MS considerably limit the utility of this technique in the analysis of mixtures such as DNA sequencing ladders or complex protein mixtures, and serious efforts to utilize ESI-MS as a sequencing tool have thus been hampered by the complexity of the resultant mass spectra.
To make ESI-MS more effective, it is desirable to decrease the charge state of electrospray generated ions. Previous approaches to charge reduction in ESI have fallen into two major categories: modification of the solution conditions (i.e., buffer, pH, salts) and utilization of gas-phase reactions within an ion trap spectrometer. Altering solution conditions does not allow predictable and controllable manipulation of the charge state for all species present in a given mixture. With conventional ion trap techniques, the cation or anion used to reduce charge has to be “trapped” along with the analyte(s). This has the practical consequence of limiting the charge reduction to a narrow m/z range of ions. Thus, previous ion trap apparatuses are limited by the nature of the ion trap to a defined m/z range and are thus not amenable to the charge reduction of large m/z ions. This is of course critical for reducing the charge of large DNA molecules.
As is evident from the foregoing, a need exists for a method of combining the simplicity of singly charged species spectra with the softness of ESI to efficiently and effectively allow high resolution mass spectral analysis of a mixture of a sample analyte solution containing a macromolecule of interest in a solvent wherein the method used is not limited to a low m/z range and wherein off-line sample purification or pre-separation is not required.
BRIEF SUMMARY OF THE INVENTION
The method of the present invention enables mass spectral analysis of a solution containing a macromolecule of interest by preparing a sample analyte solution containing the macromolecule in a solvent, discharging, with assistance of a nebulizing gas, the analyte solution through an orifice held at a high voltage in order to produce a plurality
Ebeling Daniel D.
Scalf Mark A.
Smith Lloyd M.
Westphall Michael S.
Greenlee Winner and Sullivan P.C.
Gurzo Paul M.
Lee John R.
Wisconsin Alumni Research Foundation
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