Identifying peptides at the single molecule level
The present invention relates to methods for identifying amino acids in peptides. In one embodiment, the present invention contemplates labeling the N-terminal amino acid with a first label and labeling an internal amino acid with a second label. In some embodiments, the labels are fluorescent labels. In other embodiments, the internal amino acid is lysine. In other embodiments, amino acids in peptides are identified based on the fluorescent signature for each peptide at the single molecule level.
1. A method for peptide sequencing, comprising:
a) providing a plurality of immobilized peptides on a solid support, wherein amino acids of an amino acid type of said plurality of immobilized peptides comprise a label, wherein said amino acid type is at least one of lysine, cysteine, histidine, and tyrosine;
b) contacting N-terminal amino acids of said plurality of immobilized peptides with an Edman degradation agent under conditions sufficient to remove said N-terminal amino acids of said plurality of immobilized peptides;
c) detecting said label on amino acids of said amino acid type of said plurality of immobilized peptides; and
d) repeating b) and c) one or more times to sequence said plurality of immobilized peptides.
2. The method of claim 1 , wherein said label is a fluorescent molecule.
3. The method of claim 2 , wherein said detecting comprises measuring a fluorescence intensity of said fluorescent molecule.
4. The method of claim 1 , wherein said plurality of immobilized peptides are immobilized to said solid support via internal cysteine residues.
5. The method of claim 1 , wherein said detecting comprises measuring an intensity of light emitted from said label.
6. The method of claim 1 , wherein d) comprises repeating b) and c) at least two times.
7. The method of claim 1 , wherein an N-terminal amino acid of an immobilized peptide of said plurality of immobilized peptides is of said amino acid type, wherein said immobilized peptide comprises at least one amino acid of said amino acid type separate from said N-terminal amino acid, and wherein in b) said N-terminal amino acid is removed.
8. The method of claim 1 , wherein a pattern of degradation that coincides with a reduction of signal emitted by said label is unique to at least one peptide of said plurality of immobilized peptides.
9. The method of claim 8 , wherein the pattern is compared to a proteome of an organism to identify said at least one peptide.
10. The method of claim 1 , further comprising, prior to b), contacting said plurality of immobilized peptides with an additional labeling agent under conditions sufficient to generate an additional label on amino acids of another amino acid type in said plurality of immobilized peptides.
11. The method of claim 1 , wherein all amino acids of said amino acid type in said plurality of immobilized peptides comprise said label.
12. The method of claim 1 , further comprising, prior to a), (i) providing a sample comprising a plurality of peptides, (ii) contacting said plurality of peptides with a labeling agent under conditions sufficient to generate said label on said amino acids of said amino acid type, and (iii) immobilizing said plurality of peptides on said solid support, thereby providing said plurality of immobilized peptides.
13. The method of claim 1 , wherein said amino acid type is cysteine.
14. The method of claim 1 , wherein said amino acid type is histidine.
15. The method of claim 1 , wherein said amino acid type is tyrosine.
16. The method of claim 1 , wherein said Edman degradation agent is an isothiocyanate derivative selected from the group consisting of phenyl isothiocyanate, fluorescein isothiocyanate, cyanine isothiocyanate, and rhodamine isothiocyanate.
17. The method of claim 1 , wherein in c) an absence or a reduction in signal intensity indicates that said label has been removed.