Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-08-13
2004-11-30
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S252300, C536S063000, C530S300000, C530S344000, C530S350000, C530S412000
Reexamination Certificate
active
06824981
ABSTRACT:
FIELD OF THE INVENTION
This invention is generally in the field of detection of analytes and biomolecules, and more specifically in the field of multiplex detection and analysis of analytes and biomolecules.
BACKGROUND OF THE INVENTION
Detection of molecules is an important operation in the biological and medical sciences. Such detection often requires the use of specialized label molecules, amplification of a signal, or both, because many molecules of interest are present in low quantities and do not, by themselves, produce detectable signals. Many labels, labeling systems, and signal amplification techniques have been developed. For example, nucleic acid molecules and sequences have been amplified and/or detected using polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Q&bgr; replicase (Birkenmeyer and Mushahwar,
J. Virological Methods,
35:117-126 (1991); Landegren,
Trends Genetics
9:199-202 (1993)). Proteins have been detected using antibody-based detection systems such as sandwich assays (Mailini and Maysef, “A sandwich method for enzyme immunoassay. I. Application to rat and human alpha-fetoprotein” J. Immunol. Methods 8:223-234 (1975)) and enzyme-linked immunosorbent assays (Engvall and Perlmann, “Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin” Immunochemistry 8:871-874 (1971)), and two-dimensional (2-D) gel electrophoresis (Patton,
Biotechniques
28: 944-957 (2000)). Although these techniques are useful, most have significant drawbacks and limitations. For example, radioactive labels are dangerous and difficult to handle, fluorescent labels have limited capacity for multiplex detection because of limitations on distinguishable labels, and amplification methods can be subject to spurious signal amplification. There is a need for improved detection labels and detection techniques that can detect minute quantities of specific molecules and that can be highly multiplexed.
Analysis of protein expression and presence, such as proteome profiling or proteomics, requires sensitive detection of multiple proteins. Current methods in proteome profiling suggests that there is a shortage of tools necessary for such detection (Haynes and Yates,
Proteome profiling-pitfalls and progress
. Yeast 17(2):81-87 (2000)). While the techniques of chromatography and capillary electrophoresis are amenable to proteomic studies and have seen significant development efforts (see for example, Krull et al.,
Specific applications of capillary electrochromatography to biopolymers, including proteins, nucleic acids, peptide mapping, antibodies, and so forth
. J Chromatogr A, 887:137-63 (2000), Hage,
Affinity chromatography: a review of clinical applications
. Clin Chem, 45(5):593-615 (1999), Hage et al.,
Chromatographic Immunoassays.
, Anal Chem, 73(07):198 A-205 A, (2001), Krull et al.,
Labeling reactions applicable to chromatography and electrophoresis of minute amounts of proteins
. J Chromatogr B Biomed Sci Appl, 699:173-208 (1997)), the workhorse of the industry remains two dimensional electrophoresis where the two dimensions are isoelectric focusing and molecular size. Haynes and Yates point out the significant shortcomings of the technique but discuss the utility of the method in light of such shortcomings. Hayes and Yates also discuss the techniques of Isotope Coded Affinity Tags (ICAT), LC-LC-MS/MS, and stable isotope labeling techniques (Shevchenko et al.,
Rapid ‘de novo’ peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time
-
of
-
flight mass spectrometer
. Rapid Commun Mass Spectrom 11(9):1015-1024 (1997); Oda et al.,
Accurate quantitation of protein expression and site-specific phosphorylation
. Proc Natl Acad Sci USA 96(12):6591-6596 (1999)).
Aebersold et al. (WO 00/11208) have described labels of the composition PRG-L-A, where PRG is a protein reactive group, L is a linker (that may contain isotopically distinguishable composition), and A is an affinity moiety. Aebersold et al. describes a method where the protein reactive group is used to attach the label to a protein, an affinity capture molecule is used to capture the affinity moiety, the remaining proteins are discarded, then the affinity moiety is released and the labeled proteins are detected by mass spectrometry. The method of Aebersold et al. does not involve fragmentation or other modification of the labels or proteins.
The technique of ICAT, where cysteine residues are labeled with heavy or light tags that each contain affinity moieties, in control and tester samples, has received significant interest and holds potential for protein profiling (Gygi et al.,
Quantitative analysis of complex protein mixtures using isotope-coded affinity tags
. Nat. Biotechnol. 17(10):994-999 (1999), Griffin et al.,
Quantitative proteomic analysis using a MALDI quadrupole time
-
of
-
flight mass spectrometer.
, Anal. Chem., 73:978-986 (2001)). Gygi et al. and Griffin et al. have demonstrated relative profiling of two protein samples, where the two samples are distinguished utilizing linkers containing either eight normal hydrogen or eight heavy hydrogen (deuterium) atoms. The relative concentrations of labeled proteins are determined by ratio of peaks that are separated by the corresponding 8 amu difference in the linker molecules. Current implementations have been limited to two labels. This technique does not involve fragmentation or other modification of the labels or proteins.
Mass spectrometry has been used to detect phosphorylated proteins (DeGnore and Qin,
Fragmentation of phosphopeptides in an ion trap mass spectrometer
. J. Am. Soc. Mass Spectrom. 9:1175-1188 (1998); Qin and Chait,
Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry
. Anal Chem, 69:4002-9 (1997); Annan et al.,
A multidimensional electrospray MS
-
based approach to phosphopeptide mapping
. Anal. Chem. 73:393-404 (2001)). The methods make use of a signature mass to indicate the presence of a phosphate group, for example m/z=63 and/or m/z=79 corresponding to PO
2
31
and PO
3
−
ions in negative ion mode, or the neutral loss of 98 Daltons from the parent ion indicates the loss of H
3
PO
4
from the phosphorylated peptide, indicate phosphorylated Ser, Tyr, Thr. Once phosphorylated amino acids are identified, the peptide containing the modification is sequenced by standard MS/MS techniques. There is a need for a high reliability, highly multiplexed readout system for proteomics.
The status of any living organism may be defined, at any given time in its lifetime, by the complex constellation of proteins that constitute its “proteome.”While the complete status of the proteome could be defined by listing all proteins present (including modified variants) as well as their intracellular locations and concentrations, such a task is beyond the capabilities of any current single analytical method. However, attempts have been made to define the status of a cell or tissue by identifying and measuring the relative concentrations of a small subset of proteins. For example, Conrads et al., Analytical Chemistry, 72:3349-3354 (2000), have described the use of “Accurate Mass Tags” (AMT) for proteome-wide protein identification. Conrads et al. show, for a simple organism, that a mass spectrometer of sufficient mass accuracy and resolution can be used to detect certain tryptic digest fragments from proteins. Once identified, the AMTs may be directly detected in samples by tryptic digest of the proteins, and high accuracy, high resolution mass spectrometry.
While the concept of Accurate Mass Tags is useful for protein discovery, as well as for generating peptide patterns in conventional biological experiments, it does not solve the problem of sensitivity that is at the heart of a truly useful diagnostic multi-protein assessment. A useful assessment consi
Chait Brian T.
Kershnar Eric R.
Latimer Darin R.
Lizardi Paul M.
Mattessich Martin J.
Agilix Corporation
Needle & Rosenberg PC
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