Method and system for rapid biomolecular recognition of amino acids and protein sequencing

Abstract

Methods, compositions, kits, and apparatus are provided wherein the aminoacyl-tRNA synthetase system is used to analyze amino acids. The method allows very small devices for quantitative or semi-quantitative analysis of the amino acids in samples or in sequential or complete proteolytic digestions. The methods can be readily applied to the detection and / or quantitation of one or more primary amino acids by using cognate aminoacyl-tRNA synthetase and cognate tRNA. The basis of the method is that each of the 20 synthetases and / or a tRNA specific for a different amino acid is separated spatially or differentially labeled. The reactions catalyzed by all 20 synthetases may be monitored simultaneously, or nearly simultaneously, or in parallel. Each separately positioned synthetase or tRNA will signal its cognate amino acid. The synthetase reactions can be monitored using continuous spectroscopic assays. Alternatively, since elongation factor Tu:GTP (EF-Tu:GTP) specifically binds all AA−tRNAs, the aminoacylation reactions catalyzed by the synthetases can be monitored using ligand assays. Microarrays and microsensors for amino acid analysis are provided. Additionally, amino acid analysis devices are integrated with protease digestions to produce miniaturized enzymatic sequenators capable of generating either N- or C-terminal sequence and composition data for a protein or peptide. The possibility of parallel processing of many samples in an automated manner is discussed.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. § 119(e) of U.S. Patent Application No. 60 / 224,551 filed on Aug. 10, 2000 which is incorporated herein by reference.FIELD OF THE INVENTION [0002] This invention relates to devices and methods for rapid biomolecular recognition based detection of amino acids in a sample; and, more particularly, to the application of biomolecular recognition techniques to rapid analysis of protein amino acid composition and amino acid sequences in protein. BACKGROUND [0003] Amino acids are among the most important biochemical substances in nature. Of particular interest are the 20 natural or primary protein amino acids, which are alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. In addition to being the building blocks of all proteins, the...

AI Technical Summary

Benefits of technology

[0268] An additional aspect of the present invention are methods of end-group analysis of proteins and polypeptides. Proteins that are N-terminally blocked present a challenge because they cannot be directly sequenced by Edman degradation. A blocked N-terminus is common (approximately 80-90% or eukaryotic proteins are N-terminally blocked), while C-terminal blocking is rare. One advantage of the present invention lies in rapid endgroup sequencing and analyses that may be accomplished by the methods described herein.

[0269] For example, aminopeptidases and carboxypeptidases are enzymes that release amino acids sequentially, one-at-a-time, from a protein's amino-terminus and carboxy-terminus, respectively. These enzymes are used in discontinuous kinetic assays for protein end-group sequencing. Due to the nonlinear rate of hydrolysis, those kinetic studies have been generally unsuccessful. Real-time amino acid analysis microarrays that can detect amino acids as they are released by these enzymes are thus a further aspect of this invention. These arrays are integrated with aminopeptidase or carboxypeptidase digestions, creating on-line microfluidic systems capable of generating either N-terminal or C-terminal sequence data. As amino acids are liberated by an exopeptidase, they flow from the digestion chamber through the amino acid analysis microarrays and are detected sequentially. Desirably, the digestion chamber is separated from the analysis arrays by a membrane which allows only small molecules to pass, thus protecting the channels or wells or microarrays from debris or whole proteins which could foul or plug the system. In addition, or alternatively, the protein of interest and / or the exopeptidase can be bound to the interior of the digestion chamber. The terminal sequence is generated by a computer upon analysis of the sequential amino acid detection data. If the sequence information is generated from an unidentified protein, for example, the protein can be identified by automated database searching, provided that it is one for which sequence information is available on accessible databases.

[0270] Carboxypeptidases and aminopeptidases have been used for sequencing proteins for many years (Light, A. (1967) Meth. Enzymol. 11: 426-444; Breddam and Ottesen (1987) Carlsberg Res. Comm. 52: 55-63; Royer, G. (1972) J. Biol. Chem. 218: 1807-1812). Carboxypeptidases and aminopeptidases have been used for end group protein and peptide sequencing (Martin et al. (1971) Carlsberg Res. Comm. 44: 99-102; Klarskow et al. (1989) Anal. Biochem. 180: 28-37; Thiede et al. (1995) FEBS Letts 357: 65-9; Bonetto et al. (1997) Anal. Chem. 69: 1315-1319; Bonetto et al. (1997) J. Protein Chem. 16: 371-374; Light, A. (1968) Methods Enzymol. 11: 426-444). These enzymes are ideal for removing amino acids sequentially from the N-termini of proteins (aminopeptidases) or the C-termini of proteins (carboxypeptidases). During the course of a digestion, samples are taken and analyzed later using an amino acid analyzer. Inherent in the use of these discontinuous assays is the assumption that the amino acids are being released linearly with time over the period chosen, however, the rate of cleavage is sequence dependent and varies unpredictably. When an analyte varies unpredictably, a continuous (real-time) assay is needed. A continuous assay is not possible using existing methods of amino acid analysis. This problem is solved by the continuous amino acid sensor arrays of the present method which utilize the method of amino acid analysis described herein.

[0271] Carboxypeptidases and aminopeptidases for use in sequencing reactions described herein are commercially available from numerous suppliers (e.g., Pierce Chemical Co., Rockford, Ill.). Examples of carboxypeptidases suitable for use in the subject invention for end group sequencing and end group analysis include, but are not limited to, carboxypeptidase Y, carboxypeptidase P, carboxypeptidase A, and carboxypeptidase B. Aminopeptidases, including aminopeptidases L and M, which are commercially available, are used for amino terminal sequence analysis. The exopeptidases can be immobilized using spacer arms for use in end group sequencing. Immobilized aminopeptidases and carboxypeptidases suitable for protein end group sequencing are commercially available. Mixtures of different carboxypeptidases for C-terminal sequencing and different aminopeptidases for N terminal sequencing are often used to give suitable digestion rates.

[0272] The high-throughput amino acid analysis microchips described herein are useful in the developing field of proteomics. These microarrays allow for the amino acid analysis of all proteins separated by a 2D electrophoresis gel on a single chip or plate simultaneously. In proteomics, it is especially important to determine the N-terminal and C-terminal sequence of an intact protein. End-group sequencing can be used to identify the start and stop point of a protein or gene; provides sequence information necessary for PCR cloning of the intact gene; identifies limited proteolytic products, which are common to many important regulatory mechanism; and provides a powerful method to identify proteins separated by 2D gels. Microfabricated end-group sequenators such as those described can be constructed as massively paralleled, computer controlled and integrated systems, where both N-terminal and C-terminal sequencing of many proteins can be performed on a single chip platform simultaneously. The terminal sequence tags generated can be processed on-line and the proteins identified by database searching. Among the most outstanding deficiencies in the current set of methods in protein chemistry are the ones for C-terminal sequencing. Since no sensitive and reliable method for C-terminal sequencing is available, the C-terminus of proteins is a protein region that is often not analyzed. Hence, the C-terminal sequenators of the present invention are especially useful in proteome projects.

[0273] High throughput methods for protein end group amino acid analysis methods to identify the C-terminus of proteins are further aspects of this invention. The methods for C-terminus analysis prior to the present invention are inadequate for analysis of minute quantities of protein. These inadequate methods include hydrazinolysis (Steydon, D. J. (1988) Anal. Biochem. 174: 677-686) and tritium incorporation using tritiated water after treatment of the protein with acetic anhydride to form the oxazolone (Matsuo et al. (1966) Biochem. Biophys. Res. Commun. 22: 69-74). Both methods are relatively insensitive and prone to problems. Most researchers have been forced to identify the C-terminus of a protein by peptide mapping strategies. This method is not quantitative and may miss the correct C-terminal peptide or minor but important C-terminal peptides. The present invention couples carboxypeptidase digestion with high throughput microarrays and Microsystems for rapid C-terminal analysis. The method described herein is suitable for protein end group amino acid compositional analysis, which is used to identify proteins in conjunction with sequence database searching.

Problems solved by technology

Unfortunately, current methods for amino acid analysis and protein end group analysis remain labor-intensive, slow, complicated, inaccurate, and insensitive.

Historically, the determination of amino acids in protein hydrolysates and other samples has proven to be a difficult problem.

These methods are complicated because the 20 primary amino acids do not differ from one another in any systematic way that is conducive to this analysis.

Therefore, the separation of these 20 components is difficult.

The task is further complicated by the similar structures and properties of many of the amino acids such as leucine, isoleucine, serine, and threonine, or tyrosine and phenylalanine.

This method of analysis is further hindered by the sample composition.

The presence of these compounds interferes with the analysis, since they may bind to the stationary phase during chromatography, thereby limiting the capacity or blocking the column.

In addition, most amino acids lack a strong chromophore for detection; hence amino acids need to be derivatized for detection.

All of this translates into slow, tedious, expensive and inaccurate analysis.

These complicated instruments, still in common use, are relatively insensitive (commonly nanomole detection, with lower limits of 200 to 500 picomoles of amino acids), slow, expensive and require an inordinate amount of time and effort to process a single sample.

An additional complication is the relative instability of the ninhydrin reagent.

“Within-run” and “between-run” precisions are often poor for an automated instrument.

Additionally, these systems require relatively large amounts of sample for analysis.

This requirement can be problematic in analyzing very small amounts of sample material.

The current art for amino acid analysis and protein end group analysis also presents major obstacles for the production of protein and polypeptide based products, bioresearch, and proteome projects.

The more important amino acid identification becomes the more the inadequate the existing methodology.

In short, the prior art techniques do not provide the rapid precise analysis required in today's environment, given the increased importance and demand for amino acid analysis.

Unfortunately, recent advances in rapid micro gene analysis have not been duplicated for protein or polypeptide analysis or for amino acid analysis.

This lack of development is unfortunate because, as stated above, protein and polypeptide amino acid compositional and amino acid sequence analysis are pivotal to biological research and the applications for amino acid analysis are vast.

However, due to the nonlinear rate of hydrolysis by these enzymes, kinetic assays have been unsuccessful in most cases.

However, no continuous amino acid analyzers exist.

Further, automated methods are not presently available for identifying C-terminal sequences of proteins.

C-terminal sequence tags are more specific than N-terminal sequence tags of the same length, but no reliable, sensitive method for C-terminal protein sequencing is currently available.

Images