Molecules and methods for iterative polypeptide analysis and processing
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. A method for identifying a N-terminal amino acid of a polypeptide, the method comprising:
(a) contacting the polypeptide with one or more fluorescently labeled N-terminal amino acid binding proteins (NAABs);
(b) detecting fluorescence of the NAAB that bound to the N-terminal amino acid of the polypeptide; and
(c) identifying the N-terminal amino acid of the polypeptide based on the fluorescence detected.
2. The method of claim 1 , further comprising
(d) removing the NAAB from the polypeptide; and
(e) cleaving the N-terminal amino acid of the polypeptide via Edman degradation.
3. The method of claim 2 wherein the Edman degradation comprises: reacting the N-terminal amino acid of the polypeptide with phenyl isothiocyanate (PITC) to form a PITC-derivatized N-terminal amino acid and cleaving the PITC-derivatized N-terminal amino acid using an Edman degradation enzyme.
4. The method of claim 3 wherein the Edman degradation enzyme comprises 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 cruzain; 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 85% sequence identity to the amino acid sequence of wild-type Trypanosoma cruzi cruzain of SEQ ID NO: 30.
5. The method of claim 1 , wherein the method comprises simultaneously identifying N-terminal amino acids of a plurality of polypeptides.
6. The method of claim 1 wherein the method further comprises immobilizing the polypeptide or polypeptides on a substrate prior to the contacting step (a).
7. The method of claim 6 , wherein the substrate comprises a nanogel.
8. The method of claim 1 , wherein the contacting step (a) comprises contacting the polypeptide with a single type of NAAB that selectively binds to a single type of N-terminal amino acid residue.
9. The method of claim 1 , wherein the contacting step (a) comprises contacting the polypeptide with a first type of NAAB and a second type of NAAB, wherein the first type of NAAB selectively binds to a first type of N-terminal amino acid residue and the second type of NAAB selectively binds to a second type of N-terminal amino acid residue different from the first type of N-terminal amino acid residue.
10. The method of claim 1 , wherein the fluorescently labeled NAAB comprises a fluorophore selected from the group consisting of Cy3 and Cy5.
11. The method of claim 9 , wherein the first type of NAAB is coupled to a first fluorophore and the second type of NAAB is coupled to a second fluorophore, wherein the first and second fluorophores have different fluorescence emission spectra.
12. The method of claim 2 , wherein removing the NAAB from the polypeptide comprises contacting the polypeptide with a wash buffer that causes dissociation of the NAAB bound to the N-terminal amino acid of the polypeptide.
13. The method of claim 1 , wherein the one or more NAABs 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.
14. The method of claim 2 , wherein steps (a) through (e) are repeated until the polypeptide or a fragment thereof is sequenced.
15. The method of claim 4 , wherein the remaining amino acid sequence of the Edman degradation enzyme comprises a sequence having at least 95% sequence identity to the amino acid sequence of wild-type Trypanosoma cruzi cruzain of SEQ ID NO: 30.
16. The method of claim 4 , wherein the Edman degradation enzyme comprises the amino acid sequence of SEQ ID NO: 29.
17. The method of claim 4 , wherein the Edman degradation enzyme consists of the sequence of SEQ ID NO: 29.
18. A method for cleaving the N-terminal amino group of a polypeptide, the method comprising:
(a) reacting the N-terminal amino acid of the polypeptide with phenyl isothiocyanate (PITC) to generate a PITC-derivatized N-terminal amino acid; and
(b) cleaving the modified N-terminal amino group with an Edman degradation enzyme.
19. The method of claim 18 wherein the method further comprises immobilizing the polypeptide on a substrate.
20. The method of claim 18 , wherein the Edman degradation enzyme comprises 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 wildtype Trypanosoma cruzi cruzain; 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 85% sequence identity to the amino acid sequence of wild-type Trypanosoma cruzi cruzain of SEQ ID NO: 30.