Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2002-03-29
2004-09-28
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C250S286000, C422S105000
Reexamination Certificate
active
06797945
ABSTRACT:
FIELD OF INVENTION
This invention is in the field of mass spectrometry and instrumentation for the generation of charged droplets, particularly in applications to ion sources for mass spectrometry and related analytical instruments.
BACKGROUND OF INVENTION
Over the last several decades, mass spectrometry has emerged as one of the most broadly applicable analytical tools for detection and characterization of a wide variety of molecules and ions. This is largely due to the extremely sensitive, fast and selective detection provided by mass spectrometric methods. While mass spectrometry provides a highly effective means of identifying a wide class of molecules, its use for analyzing high molecular weight compounds is hindered by problems related to generating, transmitting and detecting gas phase analyte ions of these species.
First, analysis of important biological compounds, such as oligonucleotides and oligopetides, by mass spectrometric methods is severely limited by practical difficulties related to low sample volatility and undesirable fragmentation during vaporization and ionization processes. Importantly, such fragmentation prevents identification of labile, non-covalently bound aggregates of biomolecules, such as protein—protein complexes and protein—DNA complexes, that play an important role in many biological systems including signal transduction pathways, gene regulation and transcriptional control. Second, many important biological applications require ultra-high detection sensitivity and resolution that is currently unattainable using conventional mass spectrometric techniques. As a result of these fundamental limitations, the potential for quantitative analysis of samples containing biopolymers remains largely unrealized.
For example, the analysis of complex mixtures of oligonucleotides produced in enzymatic DNA sequencing reactions is currently dominated by time-consuming and labor-intensive electrophoresis techniques that may be complicated by secondary structure. The primary limitation hindering the application mass spectrometry to the field of DNA sequencing is the limited mass range accessible for the analysis of nucleic acids. This limited mass range may be characterized as a decrease in resolution and sensitivity with an increase in ion mass. Specifically, detection sensitivity on the order of 10
−15
moles (or 6×10
8
molecules) is required in order for mass spectrometric analysis to be competitive with electrophoresis methods and detection sensitivity on the order of 10
−18
moles (or 6×10
5
molecules) is preferable. Higher resolution is be needed to resolve and correctly identify the DNA fragments in pooled mixtures particularly those resulting from Sanger sequencing reactions.
In addition to DNA sequencing applications, current mass spectrometric techniques lack the ultra high sensitivity required for many other important biomedical applications. For example, the sensitivity needed for single cell analysis of protein expression and post-translational modification patterns via mass spectrometric analysis is simply not currently available. Further, such applications of mass spectrometric analysis necessarily require cumbersome and complex separation procedures prior to mass analysis.
The ability to selectively and sensitively detect components of complex mixtures of biological compounds via mass spectrometry would tremendously aid the advancement of several important fields of scientific research. First, advances in the characterization and detection of samples containing mixtures of oligonucleotides by mass spectrometry would improve the accuracy, speed and reproducibility of DNA sequencing methodologies. In addition, such advances would eliminate problematic interferences arising from secondary structure. Second, enhanced capability for the analysis of complex protein mixtures and multi-subunit protein complexes would revolutionize the use of mass spectrometry in proteomics. Important applications include: protein identification, relative quantification of protein expression levels, identification of protein post-translational modifications, and the analysis of labile protein complexes and aggregates. Finally, advances in mass spectrometric analysis of samples containing complex mixtures of biomolecules would also provide the simultaneous characterization of both high molecular weight and low molecular weight compounds. Detection and characterization of low molecular weight compounds, such as glucose, ATP, NADH, GHT, would aid considerably in elucidating the role of these molecules in regulating a myriad of important cellular processes.
Mass spectrometric analysis involves three fundamental processes: (1) desorption and ionization of a given analyte species to generate a gas phase ion, (2) transmission of the gas phase ion to an analysis region and (3) mass analysis and detection. Although these processes are conceptually distinct, in practice each step is highly interrelated and interdependent. For example, desorption and ionization methods employed to generate gas phase analyte ions significantly influence the transmission and detection efficiencies achievable in mass spectrometry. Accordingly, a great deal of research has been directed toward developing new desorption and ionization methods suitable for the sensitive analysis of high molecular weight compounds.
Conventional ion preparation methods for mass spectrometric analysis have proven unsuitable for high molecular compounds. Vaporization by sublimation or thermal desorption is unfeasible for many high molecular weight species, such as biopolymers, because these compounds tend to have negligibly low vapor pressures. Ionization methods based on the desorption process, however, have proven more effective in generating ions from thermally labile, nonvolatile compounds. Such methods primarily consist of processes that initiate the direct emission of analyte ions from solid or liquid surfaces. Although conventional ion desorption methods, such as plasma desorption, laser desorption, fast particle bombardment and thermospray ionization, are more applicable to nonvolatile compounds, these methods have substantial problems associated with ion fragmentation and low ionization efficiencies for compounds with molecular masses greater than about 2000 Daltons.
To enhance the applicability of mass spectrometry for the analysis of samples containing large molecular weight species, two new ion preparation methods recently emerged: (1) matrix assisted laser desorption and ionization (MALDI) and (2) electrospray ionization (ESI). These methods have profoundly expanded the role of mass spectrometry for the analysis of high molecular weight compounds, such as biomolecules, by providing high ionization efficiency (ionization efficiency=ions formed/molecules consumed in analysis) applicable to a wide range of compounds with molecular weights exceeding 100,000 Daltons. In addition, MALDI and ESI are characterized as “soft” desorption and ionization techniques because they are able to both desorb into the gas phase and ionize biomolecules with substantially less fragmentation than conventional ion desorption methods.
Karas
et. al, Anal. Chem., 60, 2299-2306 (1988) and
Karas et. al, Int. J. Mass Spectrom. Ion Proc.,
78, 53-68 (1987) describe the application of MALDI as an ion source for mass spectrometry. Fenn, et. al, Science, 246, 64-71 (1989) describes the application of ESI as an ion source for mass spectrometry.
In MALDI mass spectrometry, the analyte of interest is co-crystallized with a small organic compound present in high molar excess relative to the analyte, called the matrix. The MALDI sample, containing analyte incorporated into the organic matrix, is irradiated by a short (~10 ns) pulse of UV laser radiation at a wavelength resonant with the absorption band of the matrix molecules. The rapid absorption of energy by the matrix causes it to desorb into the gas phase, carrying a portion of the analyte molecules with it. Gas phase proton transfer reactions ionize the analyte
Berggren William Travis
Scalf Mark Andrew
Smith Lloyd Michael
Westphall Michael Scott
Fernandez Kalimah
Greenlee Winner and Sullivan P.C.
Lee John R.
Wisconsin Alumni Research Foundation
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