Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Reexamination Certificate
1999-10-05
2003-05-27
Russel, Jeffrey E. (Department: 1654)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
C436S166000, C436S171000, C436S172000
Reexamination Certificate
active
06569685
ABSTRACT:
BACKGROUND
1. The Field of the Invention
This invention relates to the rapid identification of protein molecules by the systematic development for each respective type of protein molecule of a set of particular, invariant, readily-detectable distinguishing characteristics, which set of characteristics will for convenience hereinafter be referred to as a fingerprint for the corresponding type of protein molecule. The invention also relates to libraries of different protein molecules and the corresponding fingerprints therefor, as well as to systems used in the identification, or fingerprinting, of protein molecules. The present invention has particular applicability to the identification of protein molecules obtained from biological samples.
2. Background Art
There are approximately 100,000 different types of protein molecules involved in organic processes. Each protein molecule is, however, comprised of various amino acid building blocks from a group of about twenty different amino acids. Amino acids chemically connect end-to-end to form a chain that is referred to as a peptide. The amino acid building blocks in a peptide chain share as a group various of the peripheral atomic constituents of each amino acid. As a result, an amino acid in a peptide chain is not in situ a complete amino acid. Therefore, an amino acid in a peptide chain is referred to as an “amino acid residue.” A peptide chain becomes a true protein molecule only when the constituent amino acid residues have been connected, when certain amino acid residues of the peptide chain have been modified by the addition to or removal of certain types of molecules from the functional chemical groups of these amino acid residues, and when the completed chain of amino acid residues assumes a particular three-dimensional structure determined by the sequence of amino acid residues and chemical modifications thereof.
Protein molecules do not naturally maintain a one-dimensional, linear arrangement. The sequence of the amino acid residues in a protein molecule causes the molecule to assume an often complex, but characteristic three-dimensional shape. A protein molecule that has been forced out of this three-dimensional shape into a one-dimensional, linear arrangement is described as having been “linearized.”
Protein molecules are involved in virtually every biological process. Aberrant or mutant forms of protein molecules disrupt normal biological processes, thereby causing many types of diseases, including some cancers and inherited disorders, such as cystic fibrosis and hemophilia. The ability of a protein molecule to perform its intended function depends, in part, upon the sequence of amino acid residues of the protein molecule, modifications to particular amino acid residues of the protein molecule, and the three-dimensional structure of the protein molecule.
Alterations to the sequence of amino acid residues, to the modifications of particular amino acid residues, or to the three-dimensional structure of a protein molecule can change the way in which a protein molecule participates in biological processes. While many protein molecules and the functions thereof in biological processes are known, scientists continue the arduous task of isolating protein molecules, identifying the chemical composition and structure of each isolated protein molecule, and determining the functions of the protein molecule, as well as the consequences of changes in the structures of the protein molecule.
The sequence of the amino acid residues in a protein molecule, which imparts to the protein molecule a unique identity with a set of unique characteristics, is difficult to detect rapidly and reliably.
The identification of a protein molecule typically involves two steps: (1) purifying the protein molecule; and (2) characterizing the protein molecule.
In isolating or purifying protein molecules, a targeted protein molecule is separated from other, different types of protein molecules. Some current purification techniques are sensitive enough to purify an aberrant form of a protein molecule from normal protein molecules of the same type. Different purification techniques are based on the different characteristics of protein molecules, such as the weight of a protein molecule, the solubility of a protein molecule in water and other solvents, the reactivity of a protein molecule with various reagents, and the pH value at which the protein molecule is electrically neutral. The last is referred to as the isoelectric point of the protein molecule. Due to the large number of different types of protein molecules and because some types of protein molecules have very similar characteristics to other types of protein molecules, extremely sensitive purification processes are often required to isolate one type of protein molecule from others. The sensitivity with which similar types of protein molecules are separated from each other can be enhanced by combining different types of these purification techniques.
In some characterization processes, individual protein molecules are studied. When characterization processes that permit one to study individual protein molecules are employed, a single protein molecule in a sample can be separated or isolated from the other protein molecules in the sample by diluting the sample.
Since many purification techniques separate different types of protein molecules on the bases of the physical or chemical characteristics of the different types of protein molecules, these purification techniques may themselves reveal some information about the identity of a particular type of protein molecule. Once a particular type of protein molecule has been purified, it may be necessary to further characterize the purified protein molecule in order to identify the purified protein molecule. This is particularly true when attempting to characterize previously unidentified types of protein molecules, such as aberrant or mutant forms of a protein molecule.
Typically, protein molecules are further characterized by employing techniques that determine the weight of the protein molecule with increased sensitivity over techniques like gel electrophoresis, or by determining the sequence of amino acid residues that make up the protein molecule. One technique that is useful for performing both of these tasks is mass spectrometry.
In order to characterize a type of protein molecule by mass spectrometry, a purified type of protein molecule or a particular segment of a purified type of protein molecule is given positive and negative charges, or ionized, and made volatile in a mass spectrometer. The ionized, volatilized protein molecules or segments are then analyzed by the mass spectrometer. This produces a mass spectrum of the protein molecule or segment. The mass spectrum provides very precise information about the weight of the protein molecule or segment. Due to the precision with which a mass spectrometer determines the weight of protein molecules and segments of protein molecules, when a protein molecule or segment is analyzed, the information provided by mass spectrometry can be of use in inferring the sequence of amino acid residues in the protein molecule or segment. Mass spectrometers are also sensitive enough to provide information about modifications to particular amino acid residues of a protein molecule or segment. When a series of segments from a certain type of protein molecule are analyzed by mass spectrometry, the information about the sequences of and modifications to the amino acid residues of each segment can be combined to infer the sequence of and modifications to amino acid residues of an entire protein molecule.
Due to the sensitivity of mass spectrometry and the resulting ability to infer the sequences of the amino acid residues and modifications thereto of a particular type of protein molecule, the differences of aberrant or mutant forms of protein molecules from a normal protein molecule in amino acid residue sequences and amino acid residue modifications can also be inferred.
Nonetheless, mass spectrometry is a time-consu
Brent Roger
Burbulis Ian E.
Carlson Robert H.
Russel Jeffrey E.
The Molecular Sciences Institute, Inc.
TraskBritt PC
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