Molecules and methods for iterative polypeptide analysis and processing

Abstract

Reagents and methods for the digital analysis of proteins or peptides are provided. Specifically provided herein are proteins for identifying the N-terminal amino acid or N-terminal phosphorylated amino acid of a polypeptide. Also, an enzyme for use in the cleavage step of the Edman degradation reaction and a method for using this enzyme are described.

1 . An isolated, synthetic, or recombinant N-terminal amino acid binding protein (NAAB) comprising:
 (1) an amino acid sequence having a glutamate residue at a position corresponding to position 42 of wild-type  E. coli  methionine aminopeptidase (eMAP) (SEQ ID NO: 1), a tryptophan residue at a position corresponding to position 46 of wild-type eMAP, a threonine or serine residue at a position corresponding to position 56 of wild-type eMAP, an aspartate residue at a position corresponding to position 57 of wild-type eMAP, a serine residue at a position corresponding to position 58 of wild-type eMAP, a leucine residue at a position corresponding to position 59 of wild-type eMAP, a threonine or serine residue at a position corresponding to position 60 of wild-type eMAP, a histidine residue at a position corresponding to position 62 of wild-type eMAP, an asparagine residue at a position corresponding to position 63 of wild-type eMAP, a isoleucine or valine residue at a position corresponding to position 65 of wild-type eMAP, an aspartate residue at a position corresponding to position 66 of wild-type eMAP, a glycine residue at a position corresponding to position 67 of wild-type eMAP, a histidine residue at a position corresponding to position 68 of wild-type eMAP, a glycine residue at a position corresponding to position 69 of wild-type eMAP, a serine or threonine residue at a position corresponding to position 70 of wild-type eMAP, a valine residue at a position corresponding to position 81 of wild-type eMAP, an arginine residue at a position corresponding to position 101 of wild-type eMAP, a histidine residue at a position corresponding to position 177 of wild-type eMAP, and a serine or threonine residue at a position corresponding to position 221 of wild-type eMAP or   (2) an amino acid sequence having a serine residue at a position corresponding to position 13 of wild-type  E. coli  methionyl-tRNA synthetase (MetRS) (SEQ ID NO: 5); a leucine residue at a position corresponding to position 260 of wild-type  E. coli  MetRS; a glycine residue at a position corresponding to position 296 of wild-type  E. coli  MetRS; and a leucine residue at a position corresponding to position 301 of wild-type  E. coli  MetRS; wherein the remaining amino acid sequence of the NAAB comprises a sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 3.   
     
     
         2 . The NAAB of  claim 1 , wherein the remaining amino acid sequence comprises a sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of wild-type eMAP (SEQ ID NO: 1). 
     
     
         3 . The NAAB of  claim 1 , wherein the NAAB comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The isolated, synthetic, or recombinant N-terminal amino acid binding protein (NAAB) of  claim 1  comprising the amino acid sequence having a serine residue at a position corresponding to position 13 of wild-type  E. coli  methionyl-tRNA synthetase (MetRS) (SEQ ID NO: 5); a leucine residue at a position corresponding to position 260 of wild-type  E. coli  MetRS; a glycine residue at a position corresponding to position 296 of wild-type  E. coli  MetRS; and a leucine residue at a position corresponding to position 301 of wild-type  E. coli  MetRS; wherein the remaining amino acid sequence of the NAAB comprises a sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 3. 
     
     
         7 . The NAAB of  claim 6 , wherein the remaining amino acid sequence comprises a sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. 
     
     
         8 . The NAAB of  claim 6 , wherein the NAAB comprises the amino acid sequence of SEQ ID NO: 4. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . An isolated, synthetic, or recombinant N-terminal amino acid binding protein (NAAB) comprising:
 (1) an amino acid sequence having an aspartate residue at a position corresponding to position 100 of wild-type PheRS from  Thermus Thermophilus  (SEQ ID NO: 8), an asparagine residue at a position corresponding to position 142 of wild-type PheRS, a glycine residue at a position corresponding to position 143 of wild-type PheRS, a valine residue at a position corresponding to position 152 of wild-type PheRS, a glycine residue at a position corresponding to position 153 of wild-type PheRS, a lysine residue at a position corresponding to position 154 of wild-type PheRS, an aspartate residue at a position corresponding to position 165 of wild-type PheRS, an alanine residue at a position corresponding to position 205 of wild-type PheRS, an alanine residue at a position corresponding to position 212 of wild-type PheRS, a valine residue at a position corresponding to position 228 of wild-type PheRS, an asparagine residue at a position corresponding to position 229 of wild-type PheRS, a glutamate residue at a position corresponding to position 230 of wild-type PheRS, a glycine residue at a position corresponding to position 231 of wild-type PheRS, an aspartate residue at a position corresponding to position 232 of wild-type PheRS, a tyrosine residue at a position corresponding to position 234 of wild-type PheRS, a threonine residue at a position corresponding to position 257 of wild-type PheRS, a glycine residue at a position corresponding to position 287 of wild-type PheRS, an asparagine residue at a position corresponding to position 289 of wild-type PheRS, an aspartate residue at a position corresponding to position 303 of wild-type PheRS, an alanine residue at a position corresponding to position 336 of wild-type PheRS, a threonine residue at a position corresponding to position 338 of wild-type PheRS, and a glycine residue at a position corresponding to position 340 of wild-type PheRS; wherein the remaining amino acid sequence of the NAAB comprises a sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 6 or   (2) an amino acid sequence having an asparagine residue at a position corresponding to position 121 of truncated wild-type HisRS from  E. coli  (3-180) (SEQ ID NO: 9) and an alanine residue at a position corresponding to position 122 of wild-type HisRS; wherein the remaining amino acid sequence of the NAAB comprises a sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9.   
     
     
         12 . The NAAB of  claim 11 , wherein the remaining amino acid sequence comprises a sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. 
     
     
         13 . The NAAB of  claim 11 , wherein the NAAB comprises the amino acid sequence of SEQ ID NO: 7. 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The isolated, synthetic, or recombinant N-terminal amino acid binding protein (NAAB) of  claim 11  comprising the amino acid sequence having an asparagine residue at a position corresponding to position 121 of truncated wild-type HisRS from  E. coli  (3-180) (SEQ ID NO: 9) and an alanine residue at a position corresponding to position 122 of wild-type HisRS; wherein the remaining amino acid sequence of the NAAB comprises a sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9. 
     
     
         17 . The NAAB of  claim 16 , wherein the remaining amino acid sequence comprises a sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9. 
     
     
         18 . The NAAB of  claim 16 , wherein the NAAB comprises the amino acid sequence of SEQ ID NO: 10. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . A polypeptide of  claim 1 , wherein the NAAB is coupled to a fluorophore. 
     
     
         22 . The polypeptide of  claim 21  wherein the fluorophore comprises Cy3 or Cy5. 
     
     
         23 . An isolated, synthetic, or recombinant Edman degradation enzyme comprising an amino acid sequence having a glycine residue at a position corresponding to position 25 of wild-type  Trypanosoma cruzi  cruzian; a serine residue at a position corresponding to position 65; a cysteine residue at a position corresponding to position 138; and a tryptophan residue at a position corresponding to position 160; wherein the remaining amino acid sequence of the Edman degradation enzyme comprises a sequence having at least about 80% sequence identity to the amino acid sequence of wild-type  Trypanosoma cruzi  cruzian. 
     
     
         24 . The Edman degradation enzyme of  claim 23 , wherein the remaining amino acid sequence comprises a sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of wild-type  Trypanosoma cruzi  cruzian. 
     
     
         25 . The Edman degradation enzyme of  claim 23 , wherein the enzyme comprises the amino acid sequence of SEQ ID NO: 29. 
     
     
         26 . The Edman degradation enzyme of  claim 23 , wherein the enzyme consists of the sequence of SEQ ID NO: 29. 
     
     
         27 - 48 . (canceled) 
     
     
         49 . A kit for sequencing polypeptides comprising:
 one or more N-terminal amino acid binding proteins (NAABs);   the Edman degradation enzyme of  claim 23 ; and   instructions for using the NAABs and the Edman degradation enzyme for sequencing a polypeptide.   
     
     
         50 . (canceled) 
     
     
         51 . The kit of  claim 49  wherein the NAAB comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 7; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; and SEQ ID NO: 28. 
     
     
         52 - 83 . (canceled)