Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Reexamination Certificate
2000-12-12
2004-01-20
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
C436S173000, C436S501000, C436S503000
Reexamination Certificate
active
06680203
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for high information content (HIC) analysis or screening of complex biological systems using Fourier transform mass spectrometry (FTMS). The present methods are useful for analyzing complex biological mixtures containing both high molecular weight molecules (e.g., polynucleotides, proteins, polysaccharides) and low molecular weight molecules (e.g., oligonucleotides, peptides, lipids, oligosaccharides, steroid hormones, catabolic and metabolic intermediates) permit the elucidation of molecular differences between complex biological samples, and permit the identification of biologically active molecules (e.g. therapeutically active drugs, etc.).
BACKGROUND OF THE INVENTION
Mass spectrometry is an analytical technique measuring an atom's or a molecule's mass (referred to as atomic and molecular mass, respectively). Since molecular mass is the stoichiometric sum of the atomic masses for each element in the molecule, a characteristic measure is provided for each analyte having a different empirical formula.
The instrument used to measure molecular mass is known as a mass spectrometer. Typically, mass spectrometry is performed by volatilizing (in a gas phase) an analyte then ionizing an analyte and detecting signals. For most types of mass spectrometers, the detector consists of a type of electron multiplier. Ions impinging on such a detector create secondary electrons that register as some measurable current. In this respect, the FTMS instrument is uniquely different in that it measures ions indirectly and non-destructively by measuring an image current. The data generated in fine, i.e., a mass spectrum, has two coordinates: the mass-to-charge ratio scale (x-axis) and the intensity scale (y-axis).
The molecular masses of gas-phase ions, which are formed from both neutral and charged molecules, are determined based on their mass-to-charge (m/z) ratios. If further fragmentation of the gas phase ions is desired, this can be achieved by having them collide with gas molecules, so-called “collision-induced dissociation” (CID). The subfragments that are generated are then also separated by mass.
In recent years, mass spectrometry has been exploited in a variety of biological contexts, including nucleic acid sequencing, peptide sequencing and identification (Keen and Findlay, “Protein Sequencing Techniques,” in
Molecular Biology and Biotechnology
, Robert A. Meyers, ed., VCH Publishers, Inc. 1995, p. 771; Carr and Annan, “Overview of Peptide and Protein Analysis by Mass Spectrometry,” in
Current Protocols in Molecular Biology
, Ausubel et al., eds., John Wiley & Sons, Inc., 1997, 10.21); detection of in vitro and in vivo protein post-translational modification and expression (Rowley et al., 2000, Methods 20:383-397); elucidation of protein tertiary structure (Last and Robinson, 1999, Curr. Opin. Chem. Biol. 3:564-570); study of labile, non-covalently associated biomolecules (Budnik et al., 2000, Rapid Commun. Mass Spectrom.14:578-584); disease diagnosis (Bartlett and Pourfarzam, 1999, J. Inherit. Metab. Dis. 22:568-571); surveillance of environmental contamination (Scribner et al., 2000, Sci. Total Environ. 248:157-167); agricultural screening (Hau et al., 2000, J. Chromatogr. 878:77-86); and forensic applications (Hollenbeck et al., 1999, J. Forensic Sci. 44:783-788; Gaillard and Pepin, 1999, J. Chromatogr. B. Biomed. Sci. Appl. 733:181-229).
Mass spectrometry, which provides femtomolar sensitivity and accuracy better than 0.01%, has emerged as an attractive alternative to chemical methods for peptide sequencing and identification. Sensitivity of mass spectrometry has been improved by using isotopically labeled peptides and combining a nanoelectrospray ion source with a quadrupole time-of-flight tandem mass spectrometer. This approach exploits an intrinsic feature of the quadrupole time-of-flight device, affording higher sensitivity and resolution than other types of mass spectrometers (Shevchenko et al., 1997, Rapid Comm. Mass Spectrom. 11:1015-1024). Isotopic labeling of C-terminal peptide fragments, e.g., by enzymatic digestion of a protein in 1:1
16
O/
18
O water, provides a characteristic isotopic distribution for these fragments that can be readily identified (Schnolzer et al., 1996, Electrophoresis, 17:945-953); thereby revealing the amino acid sequence.
Mass spectrometry can also be used to study a protein's structure. This technology can provide accurate molecular masses for minute quantities of proteins of interest with masses up to 500,000 Daltons (“Da”). The resulting spectra also can help determine protein folding, protein self-association and other conformational changes and tertiary structure (Nguyen et al., 1995, J Chromatogr A 705:213-45). In addition, co- and post-translational modifications of proteins can be identified and mapped. This method is preferable to using chemical methods such as C-terminal sequencing, which requires relatively harsh sample treatment that can alter or destroy such protein modifications. Post-translational modifications that can be identified using mass spectrometry include phosphorylation, glycosylation, deamidation, isoaspartyl formation, and disulfide-bond formation.
Mass spectrometry has also found important applications in the study of protein-protein interactions. Target proteins can be followed in vivo to document their conformational changes, active site usage, ligand recognition, assembly into multimeric complexes (e.g., holoenzymes), and trafficking among organelles.
Fourier transform mass spectrometry (FTMS) is also known as Fourier transform ion cyclotron resonance (FTICR). The principle of molecular mass determination used in FTMS is based on a linear relationship between an ion's mass and its cyclotron frequency. In an uniform magnetic field, an ion will process about the center of the magnetic field in a periodic, circular motion known as cyclotron motion. An ensemble of ions having a particular mass-to-charge ratio (m/z) can be made to undergo cyclotron motion in-phase, producing an image current. The image current is detected between a pair of receive electrodes, producing a sine-wave signal. The Fourier transform is a mathematical deconvolution method used to separate the signals from many different m/z ensembles into a frequency, also known as mass, spectrum. Unlike other forms of mass spectrometry, FTMS is non-destructive. For a general review of FTMS, see Hendrickson and Emmett, 1999, Ann. Rev. Phys. Chem. 50:517-536. The application of FTMS to biological sciences is generally similar to other mass spectrometry applications. See, e.g., Smith et al., 1996, “The Role of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry in Biological research—New Developments and Applications” in
Mass Spectrometry in the Biological Sciences
eds. A. L. Burlingame and S. A. Carr, Humana Press, Totowa, N.J.; McLafferty, 1994, Acc. Chem. Res. 27:379-386.
A number of researchers have started evaluating the use of FTMS in the analysis of biological samples; see Jensen et al., Electrophoresis 2000 21:1372-1380; Jensen et al., Anal. Chem. 1999 71:2076-2084; Palblad et al., Rapid Comm. Mass Spec 2000, 14:1029-1034; WO 95/25281; WO 00/29987; WO00/03240; WO99/58727; WO99/57318; WO99/46047; Li et al., Anal. Chem. 1999 71:4397-4402; Penn et al., Anal. Chem. 1997; 669:2471-2477; and U.S. Pat. Nos. 6,017,093 and 4,224,031.
Analytical methods useful in drug discovery are primarily based on individual end-point observations. The targeting of specific biological interactions (e.g., receptor-ligand, substrate-enzyme) for xenobiotic intervention has been a common paradigm for mining chemical libraries. The traditional approach of choice for drug discovery by pharmaceutical, biotechnology and genomics companies is classical high throughput screening (HTS), which entails parallel screening of large chemical libraries on single targets using generally cell-free assays. Chemical libraries used in HTS are most often generated using combinatorial chemistry. Collections
Bisgaier Charles L.
Dasseux Jean-Louis H.
Newton Roger S.
Pape Michael E.
Rea Thomas J.
Esperion Therapeutics, Inc.
Gakh Yelena
Pennie & Edmonds LLP
Warden Jill
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