Differential labeling for quantitative analysis of complex protein mixtures

Abstract

The disclosed subject matter relates to a method of simultaneously identifying and determining the levels of expression of cysteine-containing proteins in normal and perturbed cells, a method for determining peptide expression levels between a first biological sample and a second biological sample, and a method for quantitative proteomic analysis of two or more peptide populations, and compounds and reagents related thereto.

1 . A method for simultaneously identifying and determining the levels of expression of cysteine-containing proteins in normal and perturbed cells, comprising:
 a) preparing a first protein sample or a first peptide sample from the normal cells;   b) subjecting the first protein sample or the first peptide sample from step a) to proteolysis;   c) reacting the proteolyzed first protein sample or the proteolyzed first peptide sample with a reagent of Formula II or III:
   Acyl-NH—X-[Epitope Tag Site] A -Y-[Protease Cleavage Site]-Z-Link  (II) 
   Acyl-NH—X-alk-O-Ph-CH 2 -Z-Link  (III) 
   where:   A is an integer from 0 to 12;   X is selected from the group consisting of an amide bond of formula —C(O)—NR—, a carbonyl of formula —C(O)—, and an amino acid sequence comprising between 0 to 10 amino acids, where R is hydrogen or lower alkyl;   Y is an amide bond of formula —C(O)—NR—, where R is hydrogen or lower alkyl, or Y is an amino acid sequence comprising between 0 to 10 amino acids;   Z is selected from the group consisting of an amide bond of formula —(CH 2 ) B —C(O)—NR—, an amide bond of formula —(CH 2 ) B —NR—C(O)—, and an amino acid sequence comprising between 0 to 3 amino acids,   where R is hydrogen or lower alkyl, and   where B is an integer from 0 to 20;   alk is straight or branched chain of alkylene comprising between 0 and 10 carbon atoms;   Ph is a phenyl group optionally substituted with one or more electron withdrawing groups ortho or para to the —CH 2 — group;   Link is selected from the group consisting of Lys-ε-iodoacetamide, Arg-δ-iodoacetamide, and Orn-δ-iodoacetamide;   Epitope Tag Site is a sequence of amino acids,   where when A is two or more, the amino acid sequence of each Epitope Tag Site can be the same or different; and   Protease Cleavage Site is an amino acid sequence of SEQ ID NO: 1 that is a cleavage site for TEV protease;   d) preparing a second protein sample or a second peptide sample from the perturbed cells;   e) subjecting the second protein sample or the second peptide sample from step d) to proteolysis;   f) reacting the proteolyzed second protein sample or the proteolyzed second peptide sample of step e) with a second reagent of Formula II or III:
   Acyl-NH—X-[Epitope Tag Site] A -Y-[Protease Cleavage Site]-Z-Link  (II) 
   Acyl-NH—X-alk-O-Ph-CH 2 -Z-Link  (III) 
   where:   A is an integer from 0 to 12;   X is selected from the group consisting of an amide bond of formula —C(O)—NR—, a carbonyl of formula —C(O)—, and an amino acid sequence comprising between 0 to 10 amino acids, where R is hydrogen or lower alkyl;   Y is an amide bond of formula —C(O)—NR—, where R is hydrogen or lower alkyl, or Y is an amino acid sequence comprising between 0 to 10 amino acids;   Z is selected from the group consisting of an amide bond of formula —(CH 2 ) B —C(O)—NR—, an amide bond of formula —(CH 2 ) B —NR—C(O)—, and an amino acid sequence comprising between 0 to 3 amino acids,   where R is hydrogen or lower alkyl, and   where B is an integer from 0 to 20;   alk is straight or branched chain of alkylene comprising between 0 and 10 carbon atoms;   Ph is a phenyl group optionally substituted with one or more electron withdrawing groups ortho or para to the —CH 2 — group;   Link is selected from the group consisting of Lys-ε-iodoacetamide, Arg-δ-iodoacetamide, and Orn-δ-iodoacetamide;   Epitope Tag Site is a sequence of amino acids,   where when A is two or more, the amino acid sequence of each Epitope Tag Site can be the same or different; and   Protease Cleavage Site is an amino acid sequence of SEQ ID NO: 1 that is a cleavage site for TEV protease,   such that the molecular weight of the first reagent and the molecular weight of the second reagent are different by an integer multiple of 14 atomic mass units;   g) combining the reacted first and second protein samples or the reacted first and second peptide sample from steps c) and f);   h) subjecting the combined protein samples or the combined peptide samples from step e) to proteolysis at a site on the protein samples or at a site on the peptide samples, the site being other than the Protease Cleavage Site;   i) subjecting the proteolyzed combined protein samples or the proteolyzed peptide samples from step f) to an affinity chromatography system comprising a second amino acid sequence attached to a solid, thereby forming bound proteins and non-bound proteins,   where the Epitope Tag Site of the reagent and the second amino acid sequence bind with high specificity to each other;   j) eluting the non-bound proteins from the affinity chromatography system;   k) subjecting the affinity chromatography system from step j) to TEV protease, thereby forming a cleaved protein mixture;   l) eluting the cleaved protein mixture from the affinity chromatography system of step k);   m) isolating the eluted protein mixture obtained from step l);   n) subjecting the eluted protein mixture from step m) to a two-dimensional liquid chromatographic separation, wherein said dimensions are selected from the group consisting of size differentiation, charge differentiation, hydrophobicity, hydrophilicity, and polarity, followed by a two-dimensional mass analysis;   o) comparing the results of step n) to:
 1) determining the ratio of amounts of compounds in the two samples, where the molecular weights thereof are separated by an integer multiple of 14 atomic mass units; and 
 2) comparing the results obtained for each compound to protein databases containing chromatographic and molecular weight correlations; 
 wherein said Z substituent in the first reagent has a molecular weight that is an integer multiple of 14 atomic mass units different than the Z substituent in the second reagent. 
   
     
     
         2 . The method of  claim 1 , wherein said Link in step c) is Lys-ε-iodoacetamide, and said Link in step f) is Orn-δ-iodoacetamide. 
     
     
         3 . The method of  claim 1 , wherein said Link in step c) is Orn-δ-iodoacetamide, and said Link in step f) is Lys-ε-iodoacetamide. 
     
     
         4 . The method of  claim 1 , wherein said reagent of step c) or step f) reacts with the reactive side chain of one or more amino acid residues of a protein in the first or second protein sample; wherein said amino acid residue is selected from the group consisting of tyrosine, cysteine, proline, and histidine. 
     
     
         5 . The method of  claim 4 , wherein said amino acid residue is a cysteine. 
     
     
         6 . The method of  claim 1 , wherein said reacting steps are carried out in a condition that is essentially free of oxygen-dependent disulfide bond formation. 
     
     
         7 . The method of  claim 6 , wherein said reacting steps are carried out in an essentially oxygen free environment. 
     
     
         8 . The method of  claim 6 , wherein said reacting steps are carried out with a reagent that reduces oxygen-dependent disulfide formation. 
     
     
         9 . A method for determining peptide expression levels between a first biological sample and a second biological sample, comprising:
 (a) providing a peptide mixture comprising first labeled peptides labeled with a first chemical group comprising a lysine residue modified with an iodoacetamide functional group on the ε-amino group of the lysine residue side chain from a first biological sample and second labeled peptides from a second biological sample labeled with a second chemical group, wherein peptides having the same amino acid sequence in the first biological sample and in the second biological sample have a predetermined mass difference resulting from a predetermined mass difference between the first chemical group and the second chemical group;   (b) calculating the weight of peptides in the peptide mixture by mass analysis;   (c) identifying a peptide pair in the peptide mixture by determining two peptides whose weight differs by the predetermined mass difference; and   (d) quantifying the abundance of each peptide in the peptide pair.   
     
     
         10 . The method of  claim 9 , wherein calculating the weight of the peptides comprises performing a primary mass analysis to produce a primary spectrum of peaks characteristic of the peptide mixture, wherein each peak corresponds to one labeled peptide in the peptide mixture. 
     
     
         11 . The method of  claim 10 , comprising performing a secondary mass analysis on each peak in order to produce a secondary spectra characteristic of the individual peptide correlated with the peak. 
     
     
         12 . The method of  claim 11 , wherein the secondary mass analysis comprises a tandem mass analytical technique selected from the group consisting of: electrospray mass analysis, fast atom bombardment mass analysis and liquid secondary ion mass analysis. 
     
     
         13 . The method of  claim 11 , comprising identifying the peptide correlated with the peak by comparing the secondary spectra with a database of known peptide spectra. 
     
     
         14 . The method of  claim 10 , wherein quantitating the abundance of each peptide comprises assessing the size of peaks in the primary spectrum to generate values representative of a relative amount of each peptide present in the peptide mixture. 
     
     
         15 . The method of  claim 14 , wherein quantitating the abundance of each peptide is performed using parallel computational methods. 
     
     
         16 . The method of  claim 10 , wherein the second chemical group comprises an ornithine residue modified with an iodoacetamide functional group on the ε-amino group of the ornithine residue side chain. 
     
     
         17 . The method of  claim 10 , wherein the first chemical group comprises  15 N and the second chemical group comprises  14 N. 
     
     
         18 . The method of  claim 10 , wherein calculating the weight of peptides comprises applying mass analytical techniques selected from the group consisting of: electron ionization mass analysis, fast atom/ion bombardment mass analysis, matrix-assisted laser desorption/ionization mass analysis and electrospray ionization mass analysis. 
     
     
         19 . The method of  claim 10 , wherein the first biological sample and the second biological sample are taken from the same starting cell population, but the first biological sample is untreated, whereas the second biological sample is treated with a test compound. 
     
     
         20 . The method of  claim 19 , wherein the starting cell population is selected from the group consisting of: plant cells, animal cells, bacterial cells and fungal cells. 
     
     
         21 . A method for quantitative proteomic analysis of two or more peptide populations, the method comprising:
 (a) differentially labeling the two or more peptide populations by labeling peptides of the two or more peptide populations with chemical groups, each chemical group associated with each of the two or more peptide populations having a predetermined mass difference from each other, and wherein at least one of the chemical groups comprises a lysine residue modified with an iodoacetamide functional group on the ε-amino group of the lysine residue side chain;   (b) combining the two or more peptide populations to form a mixed peptide population;   (c) proteolyzing the mixed peptide population to generate a collection of mixed peptide fragments of suitable size to be resolved by mass analysis;   (d) separating the collection of mixed peptide fragments by mass analysis into discrete peptide fragments while producing a primary mass spectrum with peptide peak intensities indicative of the presence of the discrete peptide fragments;   (e) analyzing the discrete peptide fragments using tandem mass analysis to generate a plurality of tandem mass spectrum characteristic of each discrete peptide fragment;   (f) comparing the tandem mass spectrum against a database of sequence-correlated mass spectra thereby determining a putative sequence identity for the tandem mass spectrum generated by the discrete peptide fragments;   (g) identifying the discrete peptide fragments derived from the differentially labeled peptide populations which are indicative of analogous peptides; and   (h) assessing the peptide peak intensities of the discrete peptide fragments derived from the analogous peptides to identify proteomic differences.   
     
     
         22 . The method for quantitative proteomic analysis of  claim 21 , wherein a sequence prediction process is used to compare the tandem mass spectrum against the database of sequence-collated mass spectra. 
     
     
         23 . The method for quantitative proteomic analysis of  claim 22 , wherein the sequence prediction process produces a plurality of sequence-correlated data files and a peak detection process is used to process and associate the sequence-correlated data files with the peptide peak intensities of the primary mass spectrum to identify the discrete peptide fragments. 
     
     
         24 . The method for quantitative proteomic analysis of  claim 23 , wherein the peak detection process operates by:
 (i) extracting information from the sequence-correlated data file corresponding to intensities for known charge states of peptide associated with the sequence-correlated mass spectrum;   (ii) identifying the highest intensity charge state of the peptide associated with the sequence-correlated mass spectrum;   (iii) identifying the peptide peak intensity in the primary mass spectrum which is associated with the highest intensity charge state of the peptide associated with the sequence-correlated mass spectrum;   (iv) performing a data filtering operation on the peptide peak intensity to remove background noise and intervening peak intensities; and   (v) performing a determination of a quantitation value to be associated with the peptide peak intensity.   
     
     
         25 . The method for quantitative proteomic analysis of  claim 22 , wherein the peak detection process wherein the peak detection process further identifies proteomic differences between analogous peptides by comparing the quantitation values for the associated discrete peptide fragments. 
     
     
         26 . The method for quantitative proteomic analysis of  claim 23 , wherein the identified proteomic differences correspond to differences in peptide concentration associated with up-regulation, down-regulation, unchanged regulation, increased peptide concentration, decreased peptide concentration, equivalent peptide concentration, peptide repression, and peptide induction.