Methods and apparatus for gel-free qualitative and quantitative proteome analysis, and uses therefore

Abstract

Methods and apparatus for qualitative and quantitative proteome analysis are provided. The methods and apparatus allow for the isolation of a subset of peptides out of complex mixtures of peptides. The isolation is based on a specific chemical and/or enzymatic alteration of one or more types of peptides. This alteration modifies the biophysical, chemical or any other biochemical property of the affected types of peptides (e.g., net electrical charge and/or hydrophobicity) in such way that the altered peptides can be separated from the unaltered peptides.

1 - 4 . (canceled)  
     
     
         5 . A method for determining the amount of at least one protein present in a sample, comprising the steps of: (a) preparing a protein peptide mixture; (b) adding to the mixture a known amount of at least one synthetic reference peptide which is differentially, isotopically labeled in comparison with its reference peptide counterpart and whereby said synthetic reference peptide and said reference peptide counterpart will be altered in step (d); (c) separating the mixture into fractions of peptides via chromatography; (d) chemically, or enzymatically, or chemically and enzymatically, altering at least one amino acid of at least one of the peptides in each fraction; (e) isolating the flagged peptides out of each fraction via chromatography, wherein the chromatography is performed with the same type of chromatography as in step (c); (f) performing mass spectrometric analysis of the flagged peptides; (g) calculating the amount of the protein present in the sample by comparing the peak highs of the synthetic reference peptide to its reference peptide; and (h) determining the identity of said reference peptides and their corresponding proteins:  
     
     
         6 . The method according to  claim 5 , wherein step (c) is preceded by one or more pre-treatment steps.  
     
     
         7 . The method according to  claim 5 , wherein the chromatographic conditions of steps (c) and (e) are the same or substantially similar.  
     
     
         8 . The method of  claim 5 , wherein the determining of the identity of the reference peptides is performed by a method selected from the group consisting of: the tandem mass spectrometric method, Post-Source Decay analysis, measurement of the mass of the peptides, and measurement of the mass of the amino-terminal peptides, in combination with database searching.  
     
     
         9 . The method of  claim 8 , wherein the determining of the identity of the reference peptides is further based on one or more of the following: (a) the presence of the altered amino acid; (b) the determination of the number of free amino groups in the reference peptides; (c) the knowledge about the cleavage specificity of the protease used to generate the protein peptide mixture; and (d) the grand average of the hydropathicity of the peptides.  
     
     
         10 . A method to determine the relative amount of at least one protein in more than one sample comprising proteins, comprising the steps of: (a) labeling the peptides present in a first sample with a first isotope; (b) labeling the peptides present in a second sample with a second isotope; (c) combining the protein peptide mixture of the first sample with the protein peptide mixture of the second sample; (d) separating the combined protein peptide mixtures into fractions of peptides via chromatography; (e) chemically, or enzymatically, or chemically and enzymatically, altering at least one amino acid in the majority of the peptides in each fraction; (f) isolating the identification peptides out of each fraction via chromatography, wherein the chromatography is performed with the same type of chromatography as in step (d); (g) performing mass spectrometric analysis of the isolated identification peptides; (h) calculating the relative amounts of the identification peptides in each sample by comparing the peak heights of the identical but differentially isotopically labeled identification peptides; and (i) determining the identity of said identification peptides and their corresponding proteins.  
     
     
         11 . The method according to  claim 10 , wherein step (d) is preceded by one or more pre-treatment steps.  
     
     
         12 . The method according to  claim 10 , wherein the chromatographic conditions of steps (d) and (f) are the same or substantially similar.  
     
     
         13 . The method of  claim 10 , wherein the determining the identity of the identification peptides is performed by a method selected from the group consisting of: the tandem mass spectrometric method, Post-Source Decay analysis, measurement of the mass of the peptides, and measurement of the mass of the amino-terminal peptides, in combination with database searching.  
     
     
         14 . The method of  claim 13 , wherein the determining of the identity of the identification peptides are further identified based on one or more of the following: (a) the determination of the number of free amino groups in the identification peptides; (b) the knowledge about the cleavage specificity of the protease used to generate the protein peptide mixture; and (c) the grand average of the hydropathicity of the peptides.  
     
     
         15 . A method for determining the amount of at least one protein present in a sample, comprising the steps of: (a) preparing a protein peptide mixture; (b) adding to the mixture a known amount of at least one synthetic reference peptide which is differentially isotopically labeled in comparison with its reference peptide counterpart whereby said synthetic reference peptide and said reference peptide counterpart will not be altered in step (d); (c) separating the mixture into fractions of peptides via chromatography; (d) chemically, or enzymatically, or chemically and enzymatically, altering at least one amino acid in the majority of the peptides in each fraction; (e) isolating the identification peptides out of each fraction via chromatography, wherein the chromatography is performed with the same type of chromatography as in step (c); (f) performing mass spectrometric analysis of the identification peptides; (g) calculating the amount of the protein present in the sample by comparing the peak highs of the synthetic reference peptide to its reference peptide counterpart and (h) determining the identity of said reference peptides and their corresponding proteins.  
     
     
         16 . The method according to  claim 15 , wherein step (c) is preceded by one or more pre-treatment steps.  
     
     
         17 . The method according to  claim 15 , wherein the chromatographic conditions of steps (c) and (e) are the same or substantially similar.  
     
     
         18 . The method of  claim 15 , wherein the determining of the identity of the reference peptides is performed by a method selected from the group consisting of: the tandem mass spectrometric method, Post-Source Decay analysis, measurement of the mass of the peptides, and measurement of the mass of the amino-terminal peptides, in combination with database searching.  
     
     
         19 . The method of  claim 18 , wherein the determining of the identity of the reference peptides is further based on one or more of the following: (a) the determination of the number of free amino groups in the reference peptides; (b) the knowledge about the cleavage specificity of the protease used to generate the protein peptide mixture; and (c) the grand average of the hydropathicity of the reference peptides.  
     
     
         20 . The method of any one of claims  5 ,  10 , or  15 , wherein the method is used to diagnose a disease or a pre-disposition to a disease.  
     
     
         21 . A system for sorting peptides comprising: a primary chromatographic column for separating a protein peptide mixture into a plurality of fractions under a defined set of conditions and whereby each fraction is subsequently subjected to an alteration of at least one amino acid to generate altered peptides and unaltered peptides and wherein the altered fractions are pooled into a set of pooled fractions, each pooled fraction comprising at least two altered fractions and a set of secondary chromatographic columns comprising a first secondary chromatographic column for separating a first pooled fraction and at least a second secondary chromatographic column arranged in parallel with the first secondary chromatographic column for separating a second pooled fraction, wherein the set of secondary chromatography columns perform isolation of the identification peptides under substantially identical conditions as the defined set of conditions, whereby there is no elution overlap between i) the identification peptides from different fractions within one pool or between pools and ii) the identification peptides and the altered peptides.  
     
     
         22 . The system of  claim 21 , further comprising an outlet to the set of second chromatography columns for collecting eluate from the first secondary chromatographic column and the second secondary chromatographic column.  
     
     
         23 . The system of  claim 22 , further comprising an analyzer connected to the outlet.  
     
     
         24 . The system of  claim 22 , further comprising a waste receptacle connected to the outlet for collecting a waste product from the set of secondary chromatography columns.  
     
     
         25 . The system of  claim 21 , further comprising a sample injector coupled to the set of secondary chromatography columns for injecting a pooled fraction into one of the first secondary column and the second secondary column.  
     
     
         26 . The system of  claim 25 , further comprising a set of sample injection valves for directing the pooled fraction from the sample injector to one of the first secondary column and the second secondary column.  
     
     
         27 . The system of  claim 21 , further comprising a solvent system for providing a solvent gradient to the set of secondary chromatographic columns.  
     
     
         28 . The system of  claim 27 , wherein the solvent system comprises a first solvent pump for providing a solvent gradient to the first secondary chromatographic column and a second solvent pump for providing a solvent gradient to the second secondary chromatographic column.  
     
     
         29 . The system of  claim 27 , wherein the solvent system comprises: a solvent pump connected to the first secondary chromatographic column and the second secondary chromatographic column; a controlled splitter system comprising a first flow rate regulator for regulating a solvent flow to the first secondary chromatographic column and a second flow rate regulator for regulating a solvent flow to the second secondary chromatographic column.  
     
     
         30 . A peptide sorting system, comprising: a first chromatographic column set comprising a first chromatographic column and a second chromatographic column arranged substantially in parallel with the first chromatographic column, a first sample injector for providing a sample to the first chromatographic column set, a first solvent system for providing a predetermined solvent gradient to the first chromatographic column set at a first predetermined time, a second chromatographic column set comprising a third chromatographic column and a fourth chromatographic column arranged in parallel with the third chromatographic column, a second sample injector for providing a sample to the second chromatographic column set, and a second solvent system for providing the solvent gradient to the second chromatographic column set at a second predetermined time subsequent to the first predetermined time.  
     
     
         31 . The system of  claim 30 , further comprising a waste receptacle connected to the outputs of the first and second chromatographic column sets for collecting a waste product the chromatographic columns.  
     
     
         32 . The system of  claim 30 , further comprising a fraction collector connected to the outputs of the first and second chromatographic column sets for collecting elute from the column.  
     
     
         33 . The system of  claim 30 , further comprising an analyzer connected to the outputs of the first and second chromatographic column.  
     
     
         34 . The system of  claim 30 , further comprising a set of inlet valves connected to the inlets of the chromatographic columns for controlling the inlets of the chromatographic columns.  
     
     
         35 . The system of  claim 34 , further comprising a set of outlet valves connected to the outlets of the chromatographic columns for directing an eluate from the columns to one of a waste receptacle, a fraction collector and an analyzer.  
     
     
         36 . The system of  claim 35 , further comprising a valve control system for controlling the set of inlet valves and the set of outlet valves.  
     
     
         37 . A method of separating peptides, comprising the steps of: providing a set of fractions of a protein peptide mixture; providing a peptide sorting system comprising a first set of parallel chromatography columns and a second set of parallel chromatography columns, loading the first set of columns with a first set of fractions of the protein peptide mixture; loading the second set of columns with a second set of fractions of the protein peptide mixture; providing a solvent gradient in the first set of columns at a first predetermined time to initialize separation of the first set of fractions; and providing the solvent gradient in the second set of columns at a second predetermined time subsequent to the first predetermined time to initialize separation of the second set of fraction.  
     
     
         38 . The method of  claim 37 , further comprising the step of directing an eluate from the first set of columns to one of a waste receptacle, a fraction collector and an analyzer.  
     
     
         39 . The method of  claim 38 , further comprising the step of directing an eluate from the second set of columns to one of a waste receptacle, a fraction collector and an analyzer.  
     
     
         40 . A method of isolating a flagged peptide from a protein peptide mixture, comprising the steps of: (a) providing a primary chromatography column for separating the protein peptide mixture; (b) injecting the protein peptide mixture into the primary chromatography column to separate the protein peptide mixture into a set of fractions under a defined set of conditions; (c) altering at least one of the fractions in the set of fractions to form a set of altered fractions, wherein an altered fraction comprises a subset of flagged peptides and a subset of unaltered peptides; (d) pooling a first altered fraction and a second altered fraction to form a first pooled fraction, wherein there is no elution overlap between i) the flagged peptides from the first and second altered fractions and ii) the flagged peptides and the unaltered peptides of said fractions; (e) pooling a third altered fraction and a fourth altered fraction to form a second pooled fraction, wherein there is no elution overlap between i) the flagged peptides from the third and fourth altered fractions and ii) the flagged peptides and the unaltered peptides of said fractions; (f) providing a first secondary chromatography column for separating a subset of flagged peptides from a subset of unaltered peptides; and (g) separating the first pooled fraction using the secondary chromatography column under the defined set of conditions to isolate the subsets of flagged peptides in the first altered fraction and the second altered fraction.  
     
     
         41 . A method of isolating an identification peptide in a protein peptide mixture, comprising the steps of: (a) providing a primary chromatography column for separating the protein peptide mixture; (b) injecting the protein peptide mixture into the primary chromatography column to separate the protein peptide mixture into a set of fractions under a defined set of conditions; (c) altering at least one of the fractions in the set of fractions to form a set of altered fractions, wherein an altered fraction comprises a subset of altered peptides and a subset of identification peptides; (d) pooling a first altered fraction and a second altered fraction to form a first pooled fraction, wherein there is no elution overlap between i) the altered peptides from the first and second altered fractions and ii) the altered peptides and the identification peptides of said fractions; (e) pooling a third altered fraction and a fourth altered fraction to form a second pooled fraction, wherein there is no elution overlap between i) the identification peptides from the third and fourth altered fractions and ii) the altered peptides and the identification peptides of said fractions; (f) providing a first secondary chromatography column for separating a subset of altered peptides from a subset of identification peptides, and (g) separating the first pooled fraction using the secondary chromatography column under the defined set of conditions to isolate the subsets of identification peptides in the first altered fraction and the second altered fraction.  
     
     
         42 . The method of  claim 40  or  41 , further comprising the step of separating the second pooled fraction using the first secondary chromatography column under the defined set of conditions to isolate the subsets of flagged or identification peptides in the third altered fraction and the fourth altered fraction.  
     
     
         43 . The method of  claim 40  or  41 , further comprising the steps of: (h) providing a second secondary chromatography column arranged substantially in parallel with the first secondary chromatography column for separating a subset of altered peptides from a subset of unaltered peptides in a fraction; and (i) separating the second pooled fraction using the second secondary chromatography column under the defined set of conditions to isolate the subsets of altered peptides in the third altered fraction and the second altered fraction.  
     
     
         44 . The method of  claim 40  or  41 , further comprising the step of directing the flagged or identification peptides to an analyzer.  
     
     
         45 . The method of  claim 44 , further comprising the step of identifying an identification or flagged peptide and its corresponding protein using the analyzer in combination with database searching.