Systems and methods for identification of MHC-I peptide epitopes
View Patent ↗Provided herein are novels systems and methods for the identification of peptides that bind to MHC-I molecules using peptide receptive MHC-I complexes.
1 . A method of purifying a peptide of interest that binds an MHC class I molecule, the method comprising:
contacting a precursor peptide-MHC-I complex (p*MHC-I) comprising a MHC Class I heavy chain, a β2 microglobulin, and a precursor peptide with a molecular chaperone, thereby forming a peptide receptive MHC-I complex comprising the MHC class I heavy chain and the β2 microglobulin;
affixing the peptide receptive MHC-I complex to a solid substrate;
contacting the peptide receptive MHC-I complex with a plurality of peptides of interest, wherein at least one of the plurality of peptides of interest binds the peptide-receptive MHC-I complex, resulting in the formation of a peptide-MHC-I complex (pMHC-I); and
removing the pMHC-I from the solid substrate, thereby purifying the peptide of interest.
2 . The method of claim 1 further comprising eluting the peptide of interest from the MHC-Class I molecule.
3 . The method of claim 2 , wherein the eluting comprises contacting the pMHC-I with a solution of acidic pH.
4 . The method of claim 3 , wherein the solution is a solution of 10% acetic acid.
5 . The method of claim 1 , further comprising identifying the peptide of interest.
6 . The method of claim 5 , wherein the peptide of interest is identified by a mass spectrometry technique.
7 . The method of claim 6 , wherein the mass spectrometry technique is liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS-MS).
8 . The method of claim 1 , wherein the peptide receptive MHC-I complex is affixed to the solid substrate via an interaction between the MHC class I heavy chain or the β2 microglobulin and the solid substrate.
9 . The method of claim 8 , wherein the MHC Class I heavy chain or the β2 microglobulin is biotinylated and the solid substrate comprises a biotin binding protein.
10 . The method of any of claim 1 , wherein the peptide receptive MHC-I complex comprises a placeholder peptide.
11 . The method of claim 1 , wherein the molecular chaperone is at a molar excess relative to the p*MHC-I.
12 . The method of claim 11 , wherein the molecular chaperone is at a ratio of more than 1:1, more than 2:1, more than 3:1, more than 4:1, more than 5:1, more than 6:1, more than 7:1, more than 8:1, more than 9:1, or more than 10:1 relative to the p*MHC-I.
13 . The method of claim 1 , wherein the p*MHC-I is at a molar excess relative to the molecular chaperone.
14 . The method of claim 1 , wherein the molecular chaperone is at a ratio of less than 1:1, less than 1:2, less than 1:5, less than 1:10, less than 1:50, less than 1:100, less than 1:500, or less than 1:1000 relative to the p*MHC-I.
15 . The method of any of claim 1 , wherein the solid substrate comprises a bead.
16 . The method of 15 , wherein the bead comprises a molecular barcode.
17 . The method of claim 15 , wherein the bead is formed into a column.
18 . The method of any of claim 1 , wherein the molecular chaperone is Tapasin Binding Protein Related (TAPBPR).
19 . The method of any of claim 1 , wherein the MHC class I heavy chains are selected from human HLA-A, human HLA-B, human HLA-C, mouse H-2D, or mouse H-2L.
20 . The method of claim 1 , wherein the MHC class I heavy chain is selected from HLA-A: 02, HLA-A: 24, or HLA-A: 68.