Single molecule peptide sequencing
Identifying proteins and peptides, and more specifically large-scale sequencing of single peptides in a mixture of diverse peptides at the single molecule level is an unmet challenge in the field of protein sequencing. Herein are methods for identifying amino acids in peptides, including peptides with one or more unnatural amino acids. In one embodiment, the N-terminal amino acid is labeled with a first label and an internal amino acid is labeled 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 of labeling of a peptide, comprising,
a) providing,
i) a peptide having at least one Cysteine amino acid, at least one Lysine amino acid, an N-terminal end, an amino acid having at least one carboxylate side group, a C-terminal end, and at least one Tryptophan amino acid, and
ii) a first compound,
iii) a second compound,
iv) a third compound,
v) a fourth compound, and
vi) a fifth compound; and
b) labeling said at least one Cysteine with said first compound,
c) labeling said at least one Lysine with said second compound,
d) labeling said N-terminal end with said third compound,
e) labeling said at least one carboxylate side group and said C-terminal end with said fourth compound; and
f) labeling said at least one Tryptophan with said fifth compound for providing a peptide having specific labels;
wherein the at least one cysteine and the at least one lysine are labeled before the N-terminal end, the at least one carboxylate side group, and the C-terminal end.
2. The method of claim 1 , wherein steps b-f are sequential in order from b-f.
3. The method of claim 1 , wherein the labeling in steps b-f are performed in one solution.
4. The method of claim 1 , wherein steps b-f are sequential in order from b-f in one solution.
5. The method of claim 1 , wherein said first compound is iodoacetatnide.
6. The method of claim 1 , wherein said second compound is 2-methylthio-2-imadazoline hydroiodide (MDI).
7. The method of claim 1 , wherein said third compound is 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl diethyl phosphate (Phos-ivDde).
8. The method of claim 1 , wherein said fourth compound is selected from the group consisting of benzylamine (BA), 3-dimethylaminopropylamine, and isobutylamine.
9. The method of claim 1 , wherein said fifth compound is 2,4-dinitrobenzenesulfenyl chloride.