Materials and methods for isolating self-antigen polypeptides
Materials and methods for isolating self-antigen polypeptides are provided.
1 . A method of isolating self-antigen polypeptides, the method comprising:
a) and a lysis buffer comprising leupeptin and pepstatin, wherein the compound is bonded to polypeptides in the cell sample by formation of an acid-labile amide bond between a dicarboxylic anhydride of the compound and primary amines of the polypeptides, thereby forming a mixed sample comprising compound bonded polypeptides,
wherein the cell sample comprises self-antigen polypeptides;
wherein the compound comprises
a first moiety comprising a methyltetrazine (mTet);
a second moiety comprising a carboxyl dimethyl maleic anhydride (CDM);
and
a linker linking the first moiety and the second moiety;
b) adding the compound bonded polypeptides in the mixed sample to an antibody coupled substrate, wherein the antibody coupled substrate comprises antibodies from serum of a patient with an autoimmune disorder bound to a bead or solid surface and wherein the compound bonded polypeptides bind to the antibody coupled substrate when the compound bonded polypeptides have an affinity to antibodies on the antibody coupled substrate, thereby forming compound bonded polypeptides bound to the antibody coupled substrate;
c) eluting the compound bonded polypeptides bound to the antibody coupled substrate by adding a solution comprising sodium dodecyl sulfate (SDS), thereby forming compound bonded self-antigen polypeptides in an eluted sample;
d) adding the compound bonded self-antigen polypeptides in the eluted sample to a trans-cyclooctene (TCO), wherein the TCO is linked to a second bead or solid surface, thereby forming self-antigen polypeptides bound to the second bead or solid surface; and
e) eluting the self-antigen polypeptides bound to the second bead or solid surface in an elution buffer having a pH more acidic than a pH of the mixed sample by reversing the acid-labile amide bond between the primary amines of the self-antigen polypeptides and the second moiety comprising a carboxyl dimethyl maleic anhydride (CDM).
2 . A method of isolating self-antigen polypeptides, the method comprising:
a) adding compound bonded polypeptides in a mixed sample to an antibody coupled substrate, wherein the compound bonded polypeptides bind to the antibody coupled substrate when the compound bonded polypeptides have an affinity to antibodies on the antibody coupled substrate, thereby forming compound bonded polypeptides bound to the antibody coupled substrate,
wherein the antibody coupled substrate comprises antibodies from serum of a patient with an autoimmune disorder bound to a bead or solid surface, and
wherein the mixed sample comprising compound bonded polypeptides is a and a lysis buffer comprising leupeptin and pepstatin, and the compound is bonded to polypeptides in the cell sample by formation of an acid-labile amide bond between a dicarboxylic anhydride of the compound and primary amines of the polypeptides,
wherein the cell sample comprises self-antigen polypeptides;
wherein the compound comprises
a first moiety comprising a methyltetrazine (mTet);
a second moiety comprising a carboxyl dimethylmaleic anhydride (CDM); and
a linker linking the first moiety and the second moiety;
b) eluting the compound bonded polypeptides bound to the antibody coupled substrate by adding a solution comprising sodium dodecyl sulfate (SDS), thereby forming compound bonded self-antigen polypeptides in an eluted sample;
c) adding the compound bonded self-antigen polypeptides in the eluted sample to a trans-cyclooctene (TCO), wherein the TCO is linked to a second bead or solid surface, thereby forming self-antigen polypeptides bound to the second bead or solid surface; and
d) eluting the self-antigen polypeptides bound to the second bead or solid surface in an elution buffer having a pH more acidic than a pH of the mixed sample by reversing the acid-labile amide bond between the primary amines of the self-antigen polypeptides and the second moiety comprising a carboxyl dimethyl maleic anhydride (CDM).
3 . The method of claim 2 , further comprising:
mixing the self-antigen polypeptides eluted in the elution buffer with a modified protease, thereby forming a mixed sample comprising self-antigen peptides and the modified protease, wherein the modified protease comprises
a protease; and
a methyltetrazine (mTet) attached to the protease, wherein the mTet forms a covalent bond with a tran-cyclooctene (TCO);
adding the modified protease in the mixed sample to a TCO, wherein the TCO is linked to the second bead or solid surface; and
eluting the self-antigen peptides.
4 . The method of claim 3 , wherein the modified protease is
(i) a modified hydrolase and the protease is a hydrolase;
(ii) a modified serine hydrolase and the protease is a serine hydrolase; or
(iii) a modified trypsin and the protease is a trypsin.
5 . The method of claim 2 , wherein the serum and cell sample are
(i) autologous samples;
(ii) allogenic samples;
(iii) the same blood sample; or
(iv) different blood samples.
6 . The method of claim 2 , wherein the self-antigen polypeptides are autoantigens or tumor-associated antigens.
7 . The method of claim 2 , wherein the linker is an inert linker.
8 . The method of claim 2 , wherein the bead or solid surface is
(i) a magnetic bead;
(ii) a bead contained within a chromatography column or a spin column; or
(iii) a protein A bead or protein G bead.
9 . The method of claim 2 , wherein the second bead or solid surface is
(i) a magnetic bead;
(ii) a bead contained within a chromatography column or a spin column; or
(iii) a porous matrix.
10 . The method of claim 2 , wherein the mixed sample has a pH greater than 7.
11 . The method of claim 10 , wherein the pH greater than 7 ranges from greater than 7 to 10, greater than 7 to 9, or 8 to 9.5.
12 . The method of claim 2 , wherein the elution buffer having a pH more acidic than a pH of the mixed sample comprises a weak organic acid selected from the group consisting of formic acid, acetic acid, and citric acid.
13 . The method of claim 2 , wherein the pH more acidic than a pH of the mixed sample ranges from 2 to less than 7, 3 to less than 7, or from 2.5 to 6.
14 . The method of claim 2 , further comprising washing the compound bonded polypeptides bound to the antibody coupled substrate to remove any unbound materials from the compound bonded polypeptides bound to the antibody coupled substrate.
15 . The method of claim 2 , further comprising washing the self-antigen polypeptides bound to the second bead or solid surface using a polar aprotic solvent to remove any unbound materials from the self-antigen polypeptides bound to the second bead or solid surface.
16 . A method of isolating self-antigen polypeptides, the method comprising:
a) adding compound bonded polypeptides in a mixed sample to an antibody coupled substrate, wherein the compound bonded polypeptides bind to the antibody coupled substrate when the compound bonded polypeptides have an affinity to antibodies on the antibody coupled substrate, thereby forming compound bonded polypeptides bound to the antibody coupled substrate,
wherein the antibody coupled substrate comprises antibodies from a biological source of a patient with an autoimmune disorder bound to a bead or solid surface, and
wherein the mixed sample comprising compound bonded polypeptides is a and a lysis buffer comprising leupeptin and pepstatin, and the compound is bonded to polypeptides in the cell sample by formation of an acid-labile amide bond between a dicarboxylic anhydride of the compound and primary amines of the polypeptides,
wherein the cell sample comprises self-antigen polypeptides;
wherein the compound comprises
a first moiety comprising a methyltetrazine (mTet);
a second moiety comprising a carboxyl dimethyl maleic anhydride (CDM); and
a linker linking the first moiety and the second moiety;
b) eluting the compound bonded polypeptides bound to the antibody coupled substrate by adding a solution comprising sodium dodecyl sulfate (SDS), thereby forming compound bonded self-antigen polypeptides in an eluted sample;
c) adding the compound bonded self-antigen polypeptides in the eluted sample to a trans-cyclooctene (TCO), wherein the TCO is linked to a second bead or solid surface, thereby forming self-antigen polypeptides bound to the second bead or solid surface; and
d) eluting the self-antigen polypeptides bound to the second bead or solid surface in an elution buffer having a pH more acidic than a pH of the mixed sample by reversing the acid-labile amide bond between the primary amines of the self-antigen polypeptides and the second moiety comprising a carboxyl dimethyl maleic anhydride (CDM).
17 . The method of claim 1 , wherein the cell sample is in a lysis buffer comprising octylphenoxypolyethoxyethanol (IGEPAL), Ethylenediaminetetraacetic acid (EDTA), Phenylmethylsulfonyl fluoride (PMSF), leupeptin, and pepstatin.
18 . The method of claim 2 , wherein the cell sample is in a lysis buffer comprising octylphenoxypolyethoxyethanol (IGEPAL), Ethylenediaminetetraacetic acid (EDTA), Phenylmethylsulfonyl fluoride (PMSF), leupeptin, and pepstatin.
19 . The method of claim 16 , wherein the cell sample is in a lysis buffer comprising octylphenoxypolyethoxyethanol (IGEPAL), Ethylenediaminetetraacetic acid (EDTA), Phenylmethylsulfonyl fluoride (PMSF), leupeptin, and pepstatin.