Capture and release based isotope tagged peptides and methods for using the same

Abstract

The invention provides non-affinity based isotope tagged peptides, chemistries for making these peptides, and methods for using these peptides. In one aspect, tags comprise a reactive site (RS) for reacting with a molecule on a protein to form a stable association with the peptide (e.g., a covalent bond) and an anchoring site (AS) group for reversibly or removably anchoring the tag to a solid phase such as a resin support. Anchoring may be direct or indirect (e.g., through a linker molecule). Preferably, the anchoring site comprises a biotin compound. Preferably, the tag comprises a mass-altering label, such as a stable isotope, such that association of the tag with the peptide can be monitored by mass spectrometry. The reagents can be used for rapid and quantitative analysis of proteins or protein function in mixtures of proteins.

1 - 18 . (canceled) 
     
     
         19 . A composition comprising a pair of tag molecules according to claim  1 , wherein each member of the pair is identical except for the mass of the isotope attached thereto. 
     
     
         20 . The composition according to  claim 19 , wherein one member of the pair comprises a heavy isotope and the other member of the pair comprises the corresponding light form of the isotope. 
     
     
         21 . A composition, comprising a reagent for mass spectrometric analysis of proteins comprising a first and second tag molecule, wherein the first tag molecule comprises a reactive site for stably associating with a protein, an isotope label, and a biotin compound linked to the tag molecule through a pH sensitive bond, the biotin compound providing an anchoring site for anchoring the tag molecule to a solid phase and the second tag molecule is identical to the first tag molecule but does not comprise an isotope label. 
     
     
         22 . A kit comprising at least one reagent according to claim  1 , and one or more of a reagent selected from the group consisting of: an activating agent for providing active groups on a protein which bind to the reactive site of the tag molecule; a solid phase; one or more agents for lysing a cell; a pH altering agent; one or more proteases; one or more cell samples or fractions thereof. 
     
     
         23 . A kit according to  claim 22 , wherein the tag molecule further comprises a peptide. 
     
     
         24 . A kit comprising at least one reagent according to  claim 21 , and one or more of a reagent selected from the group consisting of: an activating agent for providing active groups on a protein which bind to the reactive site of the tag molecule; a solid phase; one or more agents for lysing a cell; a pH altering agent; one or more proteases; one or more cell samples or fractions thereof. 
     
     
         25 . A kit according to  claim 24 , wherein the tag molecule further comprises a peptide. 
     
     
         26 . A kit comprising a plurality of tagged peptide molecules, each tagged peptide molecule comprising a peptide and a tag molecule stably associated with the protein, the tag molecule further comprising an isotope label, and a biotin compound linked to the tag molecule through a pH sensitive bond, the biotin compound providing anchoring of the tag molecule to a solid phase. 
     
     
         27 . The kit according to  claim 26 , wherein the kit comprises pairs of tagged peptides and wherein each member of a pair of tagged peptides comprises an identical peptide and each member of the pair is differentially labeled. 
     
     
         28 . The kit according to  claim 26 , comprising at least one set of tagged peptides; comprising different peptides corresponding to a single protein. 
     
     
         29 . The kit according to  claim 26 , comprising at least one set of tagged peptides comprising peptides corresponding to modified and unmodified forms of a single protein. 
     
     
         30 . The kit according to  claim 26 , comprising at least one set of tagged peptides from a first cell at a first cell state and at least one set of tagged peptides from a second cell at a second cell state. 
     
     
         31 . The kit according to  claim 30 , wherein the first cell is a normally proliferating cell and the second cell is an abnormally proliferating cell. 
     
     
         32 . The kit according to  claim 30 , wherein the first and second cells represent different stages of cancer. 
     
     
         33 . A method for identifying one or more proteins or protein functions in one or more samples containing mixtures of proteins comprising:
 reacting a sample with a first reagent according to claim  1  and a solid phase under conditions suitable to form a solid phase-isotope labeled tag molecule-protein complex;   digesting the complex with one or more proteases, thereby generating solid phase-isotope labeled tag molecule-peptide complexes and untagged peptides;   purifying the solid phase-isotope labeled tag molecule-peptide complexes;   exposing the solid phase-isotope labeled tag molecule-peptide complexes to a pH or a reducing agent which disrupts associations between the anchoring site of the tag molecule and the solid phase, thereby releasing a tagged peptide from the solid phase;   determining the mass of the tagged peptide; and   correlating the mass to the identity and/or activity of a protein.   
     
     
         34 . The method according to  claim 33 , wherein the mass-to-charge ratio of the tagged peptide is determined. 
     
     
         35 . The method according to  claim 33 , further comprising subjecting a sample comprising one or more tagged peptides to a separation step. 
     
     
         36 . The method according to  claim 35 , wherein the separation step comprises liquid chromatography. 
     
     
         37 . The method according to  claim 36 , comprising subjecting one or more tagged peptides to MS n  analysis. 
     
     
         38 . The method according to  claim 28 , further comprising reacting a second sample with a second reagent comprising an identical molecular tag as the first reagent but which is differentially labeled. 
     
     
         39 . The method according to  claim 38 , further comprising combining the two samples prior to protease digestion and generating a combined sample comprising at least one pair of tagged peptides, each member of the pair comprising identical peptides but differing in mass. 
     
     
         40 . The method according to  claim 39 , comprising determining the ratio of members of at least one tagged peptide pair in the combined sample. 
     
     
         41 . The method according to  claim 40 , further comprising generating mass spectra comprising at least one signal doublet for each peptide in the sample, the signal doublet comprising a first signal and a second signal shifted a number of known units from the first signal, wherein the known units represent the difference in molecular weight between the two members of a tagged peptide pair. 
     
     
         42 . The method according to  claim 41 , further comprising determining a signal ratio for a given peptide by relating the difference in signal intensity between the first signal and the second signal. 
     
     
         43 . The method according to  claim 33 , further comprising the step of relating mass spectra data from a tagged peptide to an amino acid sequence. 
     
     
         44 . The method according to  claim 38 , further comprising the step of relating mass spectra data from a tagged peptide to an amino acid sequence. 
     
     
         45 . The method according to  claim 33 , wherein the steps of the method are repeated, either sequentially or simultaneously, until substantially all of the proteins in a sample are detected and/or identified. 
     
     
         46 . The method according to  claim 38 , wherein the relative amounts of members of a tagged peptide pair in the two samples are determined and correlated with the abundance the protein corresponding to the peptide in the sample. 
     
     
         47 . The method according to  claim 46 , further comprising correlating the relative abundance of the protein with the state of the cells. 
     
     
         48 . The method according to  claim 47 , wherein correlating is used to diagnose a pathological condition in a patient from whom one of the cell samples was obtained. 
     
     
         49 . The method according to  claim 33 , comprising determining the quantity of a protein corresponding to the peptide in the sample. 
     
     
         50 . The method according to  claim 33 , comprising determining the site of a modification of a protein in one or more samples, by reacting sample proteins with a tag molecule comprising a reactive site which reacts with a modified residue on the protein. 
     
     
         51 . The method according to  claim 38 , comprising determining the site of a modification of a protein in one or more samples, by reacting sample proteins with a tag molecule comprising a reactive site which reacts with a modified residue on the protein. 
     
     
         52 . The method according to  claim 48 , further comprising determining the amount of modified protein in the sample. 
     
     
         53 . The method according to  claim 33 , wherein the exposing step utilizes a reducing agent comprising a phosphine. 
     
     
         54 . The method according to  claim 53 , wherein the phosphine comprises TCEP.