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 .- 5 . (canceled)
6 . A method, comprising:
a) providing a peptide immobilized on a solid support, wherein said peptide comprises at least two different types of amino acids coupled to at least two different types of labels;
b) subjecting said peptide to conditions sufficient to remove a terminal amino acid of said peptide; and
c) detecting said at least two different types of labels on said at least two different types of amino acids to sequence said peptide.
7 . The method of claim 6 , wherein said at least two different types of amino acids comprise lysine.
8 . The method of claim 6 , wherein said at least two different types of amino acids comprise a carboxylic acid side chain.
9 . The method of claim 6 , wherein said at least two different types of amino acids comprise aspartic acid.
10 . The method of claim 6 , wherein said at least two different types of amino acids comprise glutamic acid.
11 . The method of claim 6 , wherein said peptide is immobilized on said solid support via cysteine residues.
12 . The method of claim 6 , wherein said terminal amino acid is a N-terminal amino acid.
13 . The method of claim 6 , wherein said terminal amino acid is a C-terminal amino acid.
14 . The method of claim 6 , wherein said terminal amino acid of said peptide is removed by an enzyme.
15 . The method of claim 14 , wherein said enzyme comprises Edman degradation agent.
16 . The method of claim 15 , 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 6 , wherein each of said at least two different types of labels comprise a fluorescent molecule.
18 . The method of claim 17 , wherein said fluorescent molecule is an amine-reactive fluorescent molecule.
19 . The method of claim 17 , wherein said fluorescent molecule is a carboxylic acid-reactive fluorescent molecule.
20 . The method of claim 0 , wherein said detecting comprises measuring a fluorescence intensity of each of said at least two different types of labels.
21 . The method of claim 6 , wherein at least a portion of an emission spectra of each of said at least two different types of labels do not overlap with one another.
22 . The method of claim 6 , wherein, in c), a reduction in signal intensity indicates that at least one labeled amino acid of said at least two different types of amino acids coupled to said at least two different types of labels has been removed.
23 . The method of claim 6 , wherein, in c), an absence in signal intensity indicates that said at least two different types of labels have been removed.
24 . The method of claim 6 further comprising, prior to b), contacting said peptide immobilized on said solid support with an additional label under conditions sufficient to couple said additional label to another type of amino acid different from said at least two different types of amino acids.
25 . The method of claim 6 , wherein said peptide comprises at least three different types of amino acids coupled to at least three different types of labels.