Methods and reagents for biomolecule labeling, enrichment and gentle elution

Abstract

Methods and sets of non-biological reagents (elution reagents, tag isomers, tag reactive groups, crosslinkers) for single or multiplexed capture and gentle elution of biomolecules. Examples are provided using amine- and cysteine-reactive reagents for enrichment of proteins, peptides, and rare peptide modifications.

What is claimed is: 
     
         1 . A method for labeling and selective enrichment of a biomolecule in a sample, the method comprising
 (a) covalently tagging a biomolecule in the sample with at least one chemical affinity tag selected from a dimethyl piperidine- (DMP-) based chemical affinity tag or a dimethyl piperazine- (DMPZ-) based chemical affinity tag, and salts thereof, under conditions to result in a chemically tagged biomolecule   (b) selectively capturing the chemically tagged biomolecule with either
 (i) a solid support to which a chemical affinity tag epitope or analog thereof-selective antibody is attached, 
   or
 (ii) a solution comprising a chemical affinity tag epitope or analog thereof-selective antibody, and thereafter contacting the solution with a solid support capable of capturing the chemical affinity tag epitope or analog thereof-selective antibody, to selectively capture the chemically tagged biomolecule, and 
   (c) eluting the chemically tagged biomolecule by adding at least one elution reagent comprising at least one displacement molecule to the solid support to competitively elute under native conditions an intact chemically affinity tagged biomolecule.   
     
     
         2 . The method of  claim 1  where the at least one displacement molecule in step (c) is a substructure of the chemical affinity tag epitope or analog thereof. 
     
     
         3 . The method of  claim 1  where step (a) is performed on a single sample or is performed on at least two separate samples and the separate samples are combined prior to step (b) resulting in a multiplex method. 
     
     
         4 . The method of  claim 1  where the DMP-based chemical affinity tag is selected from the group consisting of 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       and structural analogs thereof, and salts thereof, where the DMP-based chemical affinity tag is optionally isotopically labeled. 
     
     
         5 . The method of  claim 1  where the chemical affinity tag further comprises a linking group and a reactive group(s), where the chemical affinity tag labels the biomolecule by at least one of amine, carboxyl, thiol, carbonyl (aldehyde/ketone), azide, alkyne, cyclic alkyne, and/or phosphine reactive chemistries. 
     
     
         6 . The method of  claim 1  further comprising, after step (c), (d) removing the at least one elution reagent by vacuum drying, desalting with dialysis, reversed phase chromatography, or size exclusion chromatography. 
     
     
         7 . The method of  claim 1  where the biomolecule is at least one of cells, proteins, peptides, glycans, steroids, nucleotides, sugars, toxins, lipids, and/or small metabolites. 
     
     
         8 . The method of  claim 1  where the at least one displacement molecule is selected from the group consisting of piperidine, cis-2,6-dimethyl piperidine, 2-S-methyl piperidine, 2-methyl piperidine, 2,2,4,4-tetramethyl piperidine, triethylamine (TEA), N,N-disopropylethylamine (DIPEA), triethylammonium bicarbonate (TEAB), triethylammonium acetate (TEAA), and combinations thereof. 
     
     
         9 . The method of  claim 1  where the chemical affinity tag epitope-selective antibody in step (b) comprises glycoforms, Fab fragments or derivatives thereof. 
     
     
         10 . The method of  claim 1  where the epitope in step (b) comprises a fragment, substructure, structural analog, or a derivative of the chemical affinity tag. 
     
     
         11 . The method of  claim 1  where the chemical affinity tag added in step (a) comprises an optional crosslinker. 
     
     
         12 . The method of  claim 1  further comprising after step (c) or  claim 6  further comprising after step (d), performing mass spectroscopy analysis on the eluted biomolecule. 
     
     
         13 . The method of  claim 1  wherein the solid support in step (b) is either a particle that is optionally magnetic, or a surface that is at least one of plastic, glass, ceramics, silicone, metal, cellulose, or gel. 
     
     
         14 . The method of  claim 1  further comprising adding at least a second chemical affinity tag to the sample, where the at least second chemical affinity tag is different from the at least one chemical affinity tag. 
     
     
         15 . The method of  claim 1  where the chemical affinity tag is either a DMP-based chemical affinity tag, having the structure 
       
         
           
           
               
               
           
         
       
       or a DMPZ-based chemical affinity tag, having the structure 
       
         
           
           
               
               
           
         
       
       where Y, if present, is a straight chain or branched C 1 -C 6  alkyl group or a straight chain or branched C 1 -C 6  alkyl ether group wherein the carbon atoms of the alkyl group or alkyl ether group each independently comprise hydrogen, deuterium or fluorine atoms;
 where R is any length linker comprised of C, N, O, H between the N-substituted ring and the reactive group(s); 
 
       and where each Z is independently hydrogen, fluorine, chlorine, bromine, iodine, an amino acid side chain, a straight chain or branched C 1 -C 6  alkyl group that may optionally contain a substituted or unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl groups each independently comprise hydrogen or fluorine atoms, a straight chain or branched C 1 -C 6  alkyl ether group that may optionally contain a substituted or unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl groups each independently comprise hydrogen or fluorine atoms or a straight chain or branched C 1 -C 6  alkoxy group that may optionally contain a substituted or unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl groups each independently comprise hydrogen or fluorine atoms. 
     
     
         16 . The method of  claim 1  using the displacement molecule in step (c) having sufficient solubility to permit competitive elution with a concentration 10 to 1,000,000,000 times the affinity binding constant of the chemical affinity tag epitope or analog thereof selective antibody affinity to the chemical affinity tag. 
     
     
         17 . The method of  claim 1  where step (c) is performed at a pH of 4 to 10. 
     
     
         18 . The method of  claim 1  where each of the dimethyl piperidine- (DMP-) based chemical affinity tag and dimethyl piperazine- (DMPZ-) based chemical affinity tag is a free base or any salt thereof. 
     
     
         19 . The method of  claim 1  using an elution reagent containing at least one buffer. 
     
     
         20 . The method of  claim 19  where the buffer is selected from the group consisting of triethylammonium bicarbonate (TEAB), triethylammonium acetate (TEAA), ((hydroxymethyl)aminomethane) (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholinopropane-1-sulfonic acid (MOPS), 1,4-piperazinediethanesulfonic acid (PIPES), bicarbonate, carbonate, N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine), N,N-(bis(2-hydroxyethyl)glycine (bicine), and combinations thereof. 
     
     
         21 . The method of  claim 1  wherein the at least one chemical affinity tag of step (a) is iodoTMT; the biomolecule in the sample is at least one S-nitrosylated peptide with S-nitrosyl groups reduced to generate free sulfhydryl groups prior to step (a), the free sulfhydryl groups reactive with iodoTMT to chemically tag the biomolecule; the chemically tagged biomolecule is selectively captured using an anti-TMT antibody resin in step (b); and the chemically tagged biomolecule is eluted in step (c). 
     
     
         22 . The method of  claim 21  wherein the sample is a plurality of blood serum samples; the iodoTMT comprises a set of isobaric chemical affinity tags used to label each of the samples; after covalently tagging the biomolecule in each of the samples in step (a), the samples are combined prior to step (b); and following step (c) of  claim 1  or step (d) of  claim 7 , performing mass spectroscopy analysis on the eluted biomolecule for a relative quantitation of peptides in a single MS analysis. 
     
     
         23 . The method of  claim 22 , wherein the sample is subjected to abundant serum protein depletion prior to covalently tagging the biomolecule in each of the samples. 
     
     
         24 . A kit comprising
 (a) a chemical affinity tag, the chemical affinity tag selected from a dimethyl piperidine- (DMP-) based chemical affinity tag or a dimethyl piperazine- (DMPZ-) based chemical affinity tag,   (b) a chemical affinity tag epitope or analog thereof-selective antibody optionally on a solid support,   (c) at least one displacement molecule selected from the group consisting of a substructure of the chemical affinity tag epitope and epitope analogs selected from the group consisting of piperidine, cis-2,6-dimethyl piperidine, 2-S-methyl piperidine, 2-methyl piperidine, 2,2,4,4-tetramethyl piperidine, triethylamine (TEA), N,N-disopropylethylamine (DIPEA), triethylammonium bicarbonate (TEAB), triethylammonium acetate (TEAA), and combinations thereof, and   (d) instructions for use of the kit to selectively label and enrich biomolecules in a sample.   
     
     
         25 . The kit of  claim 24  where the affinity tag in (a) is isotopically-labeled. 
     
     
         26 . The kit of  claim 24  where the dimethyl piperidine- (DMP-) or dimethyl piperazine- (DMPZ-) based chemical affinity tag is bound or linked to at least one biological entity or a reagent for modification of a biological entity. 
     
     
         27 . The kit of  claim 24  where the at least one displacement molecule has sufficient solubility to permit competitive elution with a concentration 10 to 1,000,000,000 times the affinity binding constant of the chemical affinity tag epitope or analog thereof-selective antibody affinity to the chemical affinity tag. 
     
     
         28 . A competitive elution buffer for labeling and selective enrichment of a biomolecule in a sample comprising
 (a) at least one displacement molecule selected from the group consisting of piperidine, cis-2,6-dimethyl piperidine, 2-S-methyl piperidine, 2-methyl piperidine, 2,2,4,4-tetramethyl piperidine, triethylamine (TEA), N,N-disopropylethylamine (DIPEA), triethylammonium bicarbonate (TEAB), triethylammonium acetate (TEAA), and combinations thereof, and   (b) at least one buffer selected from the group consisting of triethylammonium bicarbonate (TEAB), triethylammonium acetate (TEAA), ((hydroxymethyl)aminomethane) (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholinopropane-1-sulfonic acid (MOPS), 1,4-piperazinediethanesulfonic acid (PIPES), bicarbonate, carbonate, N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine), N,N-(bis(2-hydroxyethyl)glycine (bicine), and combinations thereof.