Radiant energy – Ionic separation or analysis
Reexamination Certificate
2003-06-12
2004-12-07
Wells, Nikita (Department: 2881)
Radiant energy
Ionic separation or analysis
C250S287000, C250S288000, C250S292000
Reexamination Certificate
active
06828550
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to improved methods and apparatus for mass spectrometry. In particular the invention provides methods and apparatus that allows for an accumulation of a time slice of ions to be stored in an external ion reservoir of a mass spectrometer for subsequent ion-molecule, ion-ion or dissociation reactions. The methods and apparatus of the invention can be used in the analysis of ions of macromolecules including peptides, proteins, carbohydrates, oligonucleotides and nucleic acids as well as small molecules as prepared by combinatorial or classical medicinal chemistry.
BACKGROUND OF THE INVENTION
Mass spectrometry (MS) is a powerful analytical tool for the study of molecular structure and interaction between small and large molecules. The current state of the art in MS is such that sub-femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular weight of the material may be quickly obtained, irrespective of whether the sample's molecular weight is several hundred, or in excess of a hundred thousand, atomic mass units or Daltons (Da). It has now been found that mass spectrometry can elucidate significant aspects of important biological molecules. One reason for the utility of MS as an analytical tool is the availability of a variety of different MS methods, instruments, and techniques which can provide different pieces of information about the samples.
A mass spectrometer analyzes charged molecular ions and fragment ions from sample molecules. These ions and fragment ions are then sorted based on their mass to charge ratio (m/z). A mass spectrum is produced from the abundance of these ions and fragment ions that is characteristic of every compound. In the field of biotechnology, mass spectrometry can be used to determine the structure of a biomolecule. Of particular interest is the ability of mass spectrometry to be used in determining the sequence of oligonucleotides, peptides, and oligosaccharides. Particular mass spectrometric techniques have been used to deduce the sequence of an oligonucleotide (Murray,
J. Mass Spec
., 1996, 31, 1203-1215). Mass spectrometry is also commonly used for the sequencing of peptides and proteins (Biemann,
Annu. Rev. Biochem
., 1992, 61, 977-1010).
In principle, mass spectrometers consist of at least four parts: (1) an inlet system; (2) an ion source; (3) a mass analyzer; and (4) a mass detector/ion-collection system (Skoog, D. A. and West, D. M., Principles of Instrumental Analysis, Saunders College, Philadelphia, Pa., 1980, 477-485). The inlet system permits the sample to be introduced into the ion source. Within the ion source, molecules of the sample are converted into gaseous ions. The most common methods for ionization are electron impact (EI), electrospray ionization (ESI), chemical ionization (CI) and matrix-assisted laser desorption ionization (MALDI). A mass analyzer resolves the ions based on mass-to-charge ratios. Mass analyzers can be based on magnetic means (sector), time-of-flight, quadrupole and Fourier transform mass spectrometry (FTMS). A mass detector collects the ions as they pass through the detector and records the signal. Each ion source can potentially be combined with each type of mass analyzer to generate a wide variety of mass spectrometers.
Mass spectrometry ion sources are well-known in the art. Two commonly used ionization methods are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) (Smith et al., Anal. Chem., 1990, 62, 882-899; Snyder, in Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, American Chemical Society, Washington, D.C., 1996; and Cole, in Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, Wiley, N.Y., 1997).
ESI is a gentle ionization method that results in no significant molecular fragmentation and preserves even weakly bound complexes between biopolymers and other molecules so that they are detected intact with mass spectrometry. ESI produces highly charged droplets of the sample being studied by gently nebulizing a solution of the sample in a neutral solvent in the presence of a very strong electrostatic field. This results in the generation of highly charged droplets that shrink due to evaporation of the neutral solvent and ultimately lead to a “coulombic explosion” that affords multiply charged ions of the sample material, typically via proton addition or abstraction, under mild conditions. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight biopolymers such as proteins and nucleic acids greater than 10 kDa in mass, for it affords a distribution of multiply-charged molecules of the sample biopolymer without causing any significant amount of fragmentation. The fact that several peaks are observed from one sample, due to the formation of ions with different charges, contributes to the accuracy of ESI-MS when determining the molecular weight of the biopolymer because each observed peak provides an independent means for calculation of the molecular weight of the sample. Averaging the multiple readings of molecular weight so obtained from a single ESI-mass spectrum affords an estimate of molecular weight that is much more precise than would be obtained if a single molecular ion peak were to be provided by the mass spectrometer. Further adding to the flexibility of ESI-MS is the capability of obtaining measurements in either the positive or negative ionization modes.
ESI-MS has been used to study biochemical interactions of biopolymers such as enzymes, proteins and macromolecules such as oligonucleotides and nucleic acids and carbohydrates and their interactions with their ligands, receptors, substrates or inhibitors (Bowers et al.,
Journal of Physical Chemistry
, 1996, 100, 12897-12910; Burlingame et al.,
J. Anal. Chem
., 1998, 70, 647R-716R; Biemann,
Ann. Rev. Biochem
., 1992, 61, 977-1010; and Crain et al.,
Curr. Opin. Biotechnol
., 1998, 9, 25-34). While interactions that lead to covalent modification of biopolymers have been studied for some time, one of the most significant developments in the field has been the observation, under appropriate solution conditions and analyte concentrations, of specific non-covalently associated macromolecular complexes that have been promoted into the gas-phase intact (Loo,
Mass Spectrometry Reviews
, 1997, 16, 1-23; Smith et al.,
Chemical Society Reviews
, 1997, 26, 191-202; Ens et al., Standing and Chernushevich, Eds.,
New Methods for the Study of Biomolecular Complexes, Proceedings of the NATO Advanced Research Workshop
, held Jun. 16-20 1996, in Alberta, Canada, in NATO ASI Ser., Ser. C, 1998, 510, Kluwer, Dordrecht, Netherlands).
A variety of non-covalent complexes of biomolecules have been studied using ESI-MS and reported in the literature (Loo, Bioconjugate Chemistry, 1995, 6, 644-665; Smith et al., J. Biol. Mass Spectrom. 1993, 22, 493-501; Li et al., J. Am. Chem. Soc., 1993, 115, 8409-8413). These include the peptide-protein complexes (Busman et al., Rapid Commun. Mass Spectrom., 1994, 8, 211-216; Loo et al., Biol. Mass Spectrom., 1994, 23, 6-12; Anderegg and Wagner, J. Am. Chem. Soc., 1995, 117, 1374-1377; Baczynskyj et al., Rapid Commun. Mass Spectrom., 1994, 8, 280-286), interactions of polypeptides and metals (Loo et al., J. Am. Soc. Mass Spectrom., 1994, 5, 959-965; Hu and Loo, J. Mass Spectrom., 1995, 30, 1076-1079; Witkowska et al., J. Am. Chem. Soc., 1995, 117, 3319-3324; Lane et al., J. Cell Biol., 1994, 125, 929-943), and protein-small molecule complexes (Ganem and Henion, ChemTracts-Org. Chem., 1993, 6, 1-22; Henion et al., Ther. Drug Monit., 1993, 15, 563-569; Ganguly et al.,
Tetrahedron
, 1993, 49, 7985-7996, Baca and Kent, J. Am. Chem. Soc., 1992, 114, 3992-3993). Further, the study of the quaternary structure of multimeric proteins (Baca and Kent, J. Am. Chem. Soc., 1992, 114, 3992-3993; Light-Wahl et al., J. Am. Chem. Soc., 1994
Griffey Richard
Hofstadler Steven
Cutler Wilmer
Hashmi Zia R.
ISIS Pharmaceuticals Inc.
Pickering Hale and Dorr LLP
Wells Nikita
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