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.

1 - 102 . (canceled)  
     
     
         103 . A method for determining the terminal amino acid sequence of a polypeptide, the method comprising: 
 contacting the polypeptide with an exopeptidase under reaction conditions wherein the exopeptidase removes at least one primary amino acid residue from a terminal portion of the polypeptide to release the corresponding primary amino acid; and    monitoring over time the release of each released corresponding primary amino acid to determine the order of the release of each released amino acid by    1) contacting each released amino acid with a plurality of aminoacyl tRNA synthetases, including an aminoacyl tRNA synthetase cognate for each released corresponding primary amino acid, wherein each member synthetase of the plurality differs from other member synthetases of the plurality according to the cognate amino acid thereof,    wherein contacting each released corresponding primary amino acid with a plurality of aminoacyl tRNA synthetases is under reaction conditions capable of forming a first product with the released corresponding primary amino acid, wherein the first product is selected from the group consisting of the aminoacyl-tRNA synthetase:amino acid AMP complex of the released corresponding primary amino acid, inorganic pyrophosphate, the aminoacyl-tRNA corresponding to the released corresponding primary amino acid, and adenosine monophosphate (AMP), to thereby form the first product; and    2) specifically detecting the first product for each released corresponding primary amino acid;    whereby the terminal primary amino acid residue or sequence of the polypeptide is determined according to the order of release.    
     
     
         104 . The method of  claim 103 , wherein the exopeptidase is a carboxypeptidase, the terminal portion is a C-terminal portion, and wherein the C-terminal residue of the polypeptide is determined.  
     
     
         105 . The method of  claim 104 , whereby the C-terminal amino acid sequence of the polypeptide is determined.  
     
     
         106 . The method of  claim 103 , wherein the exopeptidase is an aminopeptidase, the terminal portion is a N-terminal portion, and wherein the N-terminal residue of the polypeptide is determined.  
     
     
         107 . The method of  claim 106 , wherein the N-terminal amino acid sequence of the polypeptide is determined.  
     
     
         108 . The method of  claim 103 , wherein the monitoring is continuous.  
     
     
         109 . The method of  claim 103 , wherein the monitoring is discontinuous.  
     
     
         110 . The method of  claim 103 , wherein the first product for each released corresponding primary amino acid is inorganic pyrophosphate.  
     
     
         111 . The method of  claim 103 , wherein the first product for each released corresponding primary amino acid is AMP.  
     
     
         112 . The method of  claim 103 , wherein the first product for each released corresponding primary amino acid is the aminoacyl tRNA synthetase-aminoacyl-adenosine monophosphate complex of the released corresponding primary amino acid.  
     
     
         113 . The method of  claim 103 , wherein the first product for each released corresponding primary amino acid is the aminoacyl-tRNA of the released corresponding primary amino acid.  
     
     
         114 . The method of  claim 103 , wherein the terminal amino acid sequence of the polypeptide is determined.  
     
     
         115 . The method of  claim 114 , wherein the terminal amino acid sequence is determined for about five residues of the polypeptide.  
     
     
         116 . The method of  claim 103 , wherein the plurality of amino acyl tRNA synthetases comprises members cognate to each of the 20 primary amino acids.  
     
     
         117 . The method of  claim 103 , wherein the members of the plurality of aminoacyl tRNA synthetases are spatially resolved with each member at a known locus, and wherein each released corresponding amino acid is specifically detected according to the known locus of the aminoacyl tRNA synthetase cognate to the released corresponding amino acid.  
     
     
         118 . The method of  claim 103 , wherein each member of the plurality of aminoacyl tRNA synthetases is immobilized on a solid support.  
     
     
         119 . The method of  claim 117 , wherein each member of the plurality of aminoacyl tRNA synthetases is immobilized on a solid support.  
     
     
         120 . The method of  claim 103 , wherein each member of the plurality of first products is labeled and the detecting is by means of detecting the label.  
     
     
         121 . The method of  claim 117 , wherein each member of the plurality of first products is labeled and the detecting is by means of detecting the label.  
     
     
         122 . The method of  claim 103 , wherein each released corresponding primary amino acid is contacted with a plurality of tRNAs, wherein each member tRNA of the plurality of tRNAs differs from other member tRNAs of the plurality of tRNAs according to the cognate primary amino acid thereof and wherein the plurality of tRNAs has a member cognate for each released corresponding primary amino acid and wherein each member tRNA is spatially located separate from other member tRNAs at a known locus on a spatial array, to form each first product at a known location of the array; and 
 detecting each first product and identifying each released corresponding primary amino acid according to the known location of each first product, and wherein the first product is selected from the group consisting of inorganic pyrophosphate, adenosine monophosphate, and the aminoacyl-tRNA complex of the released corresponding primary amino acid.    
     
     
         123 . The method of  claim 122 , wherein each member tRNA is immobilized on a solid support and each first product aminoacyl-tRNA complex is thereby immobilized on the solid support.  
     
     
         124 . The method of  claim 122 , wherein each member tRNA is labeled and the label is used to detect the first product.  
     
     
         125 . The method of  claim 113 , wherein each first product is detected by contacting the aminoacyl tRNA with an elongation factor binary complex with GTP or a GTP analog to form a ternary complex and by detecting the ternary complex.  
     
     
         126 . The method of  claim 125 , wherein the elongation factor is elongation factor Tu or elongation factor 1A.  
     
     
         127 . The method of  claim 126 , wherein the GTP analog is a nonhydrolyzable analog.  
     
     
         128 . The method of  claim 125 , wherein the elongation factor is labeled.  
     
     
         129 . The method of  claim 103 , wherein the exopeptidase is immobilized on a solid support and the exopeptidase is an aminopeptidase.  
     
     
         130 . The method of  claim 103 , wherein the exopeptidase is immobilized on a solid support and the exopeptidase is a carboxypeptidase.  
     
     
         131 . The method of  claim 103 , wherein the contacting of the polypeptide with the exopeptidase takes place is separated from the aminoacyl tRNA synthetases and reaction products by a molecular sieve through which each released corresponding primary amino acid can pass and which restricts the passage of the exopeptidase and polypeptide.  
     
     
         132 . The method of  claim 131 , wherein the molecular sieve is a membrane or microdialysis probe.  
     
     
         133 . The method of  claim 131 , wherein the molecular sieve is an ultrafiltration membrane.  
     
     
         134 . The method of  claim 103 , wherein the exopeptidase and polypeptide are contacted in a gel.  
     
     
         135 . The method of  claim 103 , the polypeptide or exopeptidase are bound to the interior of the digestion chamber.  
     
     
         136 . The method of  claim 103 , wherein the polypeptide is immobilized.  
     
     
         137 . The method of  claim 103 , wherein the contacting takes place within a chip enzymatic nanosequenator.  
     
     
         138 . The method of  claim 103 , wherein the detecting is quantitative and the amount of the released corresponding primary amino acid is thereby determined.  
     
     
         139 . The method of  claim 103 , wherein the contacting of the polypeptide with the exopeptidase takes is separated from the plurality of aminoacyl tRNA synthetases by a molecular sieve through which compounds greater that about 6 kDa cannot pass.  
     
     
         140 . The method of  claim 122 , wherein each member tRNA of the plurality is uniquely labeled.  
     
     
         141 . The method of  claim 123 , wherein the solid support is a transducer and the immobilization creates a biosensor for detection of the aminoacyl tRNA.  
     
     
         142 . The method of  claim 141 , wherein the transducer is an electrode, an optical waveguide, optical partical, thermistor or piezoelectric crystal.  
     
     
         143 . The method of  claim 125 , wherein each elongation factor GTP complex or elongation GTP analog complex is immobilized on a solid support.  
     
     
         144 . The method of  claim 125 , wherein the elongation factor GTP complex is immobilized on a transducer and the immobilization creates a biosensor for the detection of the ternary complex of the elongation factor:GTP-aminoacyl-tRNA.  
     
     
         145 . The method of  claim 144 , wherein the transducer is an electrode, an planar waveguide, an optical fiber, a thermistor or a piezoelectric crystal.  
     
     
         146 . The method of  claim 122 , wherein each member of the spatial array is located on a biosensor surface.  
     
     
         147 . The method of  claim 146 , wherein the biosensor is a planar waveguide, optical fiber, electrode, or surface plasmon resonance based biosensor.  
     
     
         148 . The method of  claim 120 , wherein the label is a fluorescent label.  
     
     
         149 . The method of  claim 120 , wherein the label is a chromophore, a nanoparticle, a metal, an enzyme, an electrogenic label, biotin or a radiolabel.  
     
     
         150 . The method of  claim 125 , wherein the elongation factor binary complex with GTP is labeled and the detecting of the ternary complex detects the label.  
     
     
         151 . The method of  claim 125 , wherein a ternary complex probe is used in the detecting of the ternary complex.  
     
     
         152 . The method of  claim 151 , wherein the ternary complex probe is an antibody or fragment thereof specific for the ternary complex or components thereof.  
     
     
         153 . The method of  claim 151 , wherein the ternary complex probe is a nucleic acid.  
     
     
         154 . The method of  claim 151 , wherein the ternary complex probe is labeled and the detecting detects the label.  
     
     
         155 . The method of  claim 103 , wherein each released corresponding primary amino acids is selected from the group consisting of alanine, arginine asparagines, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine with the proviso that when the primary amino acid in the sample is arginine, glutamic acid, or glutamine the aminoacyl tRNA synthetase cognate to the primary amino acid is prebound to the tRNA cognate to the primary amino acid.  
     
     
         156 . A system for the detection of at least one primary amino acid released from the N- or C-terminal end of a polypeptide by aminopeptidase or carboxypeptidase digestion, respectively, wherein the system comprises: 
 an array of a plurality of spatially separated enzymatically active aminoacyl tRNA synthetases or of a plurality of spatially separated tRNAs cognate to a plurality of the primary amino acids, wherein each member of the array is at a known locus on the array, and wherein the plurality includes member aminoacyl tRNA synthetases cognate to each released primary amino acid;    means for contacting each released primary amino acid with the spatially separated synthetases or spatially separated tRNAs of the array under reaction conditions capable of forming a first product with each released primary amino acid, wherein the first product is selected from the group consisting of the aminoacyl-tRNA synthetase:amino acid AMP complex of the released primary amino acid, inorganic pyrophosphate, the aminoacyl-tRNA corresponding to the released primary amino acid, and AMP; and    means for specifically monitoring over time the amount of the first product formed for each released primary amino acid.    
     
     
         157 . The system of  claim 156 , wherein the spatially separated aminoacyl tRNA synthetases or spatially separated tRNAs of the array collectively provide an aminoacyl tRNA synthetase or tRNA for each of the 20 primary amino acids.  
     
     
         158 . The system of  claim 156 , wherein the array is formatted as a microparticle, microbead, microsphere, microspot, microwell, or microfluidic array.  
     
     
         159 . The system of  claim 156 , wherein the array is formatted as a biosensor array in which the spatially separated enzymatically active aminoacyl tRNA synthetases or the spatially separated tRNAs of the array are each immobilized on a transducer.  
     
     
         160 . The system of  claim 159 , wherein the transducer is a planar waveguide, optical fiber, electrode or optical particle.  
     
     
         161 . The system of  claim 156 , wherein the monitoring is continuous.  
     
     
         162 . The system of  claim 156 , wherein the monitoring provides a plurality of signals indicating the identity and amount for each first product at different times, and wherein the system further comprises 
 a computer to receive the plurality of signals and which is programmed to plot the amount of each amino acid detected versus time.    
     
     
         163 . The system of  claim 156 , wherein the monitoring provides a plurality of signals indicating the identity and amount for each first product at a time of monitoring, and wherein the system further comprises 
 a microprocessor or computer to receive the plurality of signals and which is programmed to generate the terminal amino acid sequence of the polypeptide according to said signals.    
     
     
         164 . The system of  claim 163 , further comprising an electronic database of protein or polypeptide amino acid sequences wherein the computer or microprocessor is electronically linked to the electronic database to automatically provide the name of a protein or polypeptide from the database having the same terminal amino acid sequence as the generated terminal amino acid sequence.  
     
     
         165 . A method of measuring the amount of a polypeptide in a sample, said method comprising: 
 contacting the sample with an exopeptidase under reaction conditions wherein the exopeptidase stoichiometrically removes at least one primary amino acid residue from a terminal portion of the polypeptide in the sample to stoichiometrically release the corresponding primary amino acid; and    monitoring over time the release of each released corresponding primary amino acid to determine the order of the release of each released amino acid by 
 1) contacting each released amino acid with a plurality of aminoacyl tRNA synthetases, including an aminoacyl tRNA synthetase cognate for each released corresponding primary amino acid, wherein each member synthetase of the plurality differs from other member synthetases of the plurality according to the cognate amino acid thereof,  
 wherein contacting each released corresponding primary amino acid with a plurality of aminoacyl tRNA synthetases is under reaction conditions capable of forming a first product with the released corresponding primary amino acid, wherein the first product is selected from the group consisting of the aminoacyl-tRNA synthetase:amino acid AMP complex of the released corresponding primary amino acid, inorganic pyrophosphate, the aminoacyl-tRNA corresponding to the released corresponding primary amino acid, and adenosine monophosphate (AMP), to thereby form the first product; and  
 2) specifically and quantitatively detecting the first product for each released corresponding primary amino acid to determine the number of molecules or molecular concentration of each first product formed;  
   and from the amount of each first product formed determining the amount of the polypeptide in the sample.    
     
     
         166 . The method of  claim 165 , wherein the released corresponding primary amino acid is the terminal amino acid of the polypeptide.  
     
     
         167 . The method of  claim 165 , wherein the exopeptidase is a carboxypeptidase, the terminal portion of the polypeptide is the C-terminus, and the corresponding primary amino acid is the C-terminal amino acid of the polypeptide.  
     
     
         168 . The method of  claim 165 , wherein the exopeptidase is an aminopeptidase, the terminal portion of the polypeptide is the N-terminus, and the corresponding primary amino acid is the N-terminal amino acid of the polypeptide.