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

• US 6,908,740 B2
• Filed: 03/21/2003
• Issued: 06/21/2005
• Est. Priority Date: 03/22/2001
• Status: Expired due to Term

• Current Assignee: MyCartis NV
• Original Assignee: Flanders Interuniversity for Biotechnology
• Inventors: Vandekerckhove, Joel, Gevaert, Kris
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US10/394,980
• Publication Number:
  US 20040005633A1
• Time in Patent Office: 823 Days
• Field of Search: 435/7.1, 435/23, 435/24, 435/4, 435/803, 435/287.2, 530/417, 530/300
• US Class Current: 435/7.1
• CPC Class Codes: B01D 15/1871 placed in series
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: G01N 2030/8411 Intermediate storage of effluent, including condensation on surface
• CPC Class Codes: G01N 30/461 with serial coupling of separation columns
• CPC Class Codes: G01N 30/467 all columns being identical
• CPC Class Codes: G01N 30/468 involving switching between different column configurations
• CPC Class Codes: G01N 30/7233 interfaced to liquid or supercritical fluid chromatograph interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04
• CPC Class Codes: G01N 30/80 Fraction collectors
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding

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.

Claims

1. A method for the isolation of a subset of peptides out of a protein peptide mixture, comprising the steps of:

(a) separating the protein peptide mixture into fractions of peptides via chromatography;

(b) chemically, or enzymatically, or chemically and enzymatically, altering at least one amino acid of at least one of the peptides in each fraction, thereby generating a subset of altered peptides; and

(c) isolating said altered or flagged peptides out of each fraction via chromatography, wherein the chromatography of steps (a) and (c) is performed with the same type of chromatography.

2. 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 of at least one of the peptides in each fraction;

(f) isolating the flagged 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 flagged peptides;

(h) calculating the relative amounts of the flagged peptides in each sample by comparing the peak heights of the identical but differentially, isotopically labelled flagged peptides, and (i) determining the identity of said flagged peptides and their corresponding proteins.

3. A method for the isolation of a subset of peptides out of a protein peptide mixture, comprising the steps of:

(a) separating the protein peptide mixture into fractions of peptides via chromatography;

(b) chemically, or enzymatically, or chemically and enzymatically, altering at least one amino acid in the majority of the peptides in each fraction, thereby generating a subset of unaltered peptides; and

(c) isolating said unaltered or so called identification peptides out of each fraction via chromatography, wherein the chromatography of steps (a) and (c) is performed with the same type of chromatography.

4. 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 flagged 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 flagged peptides under substantially identical conditions as the defined set of conditions, whereby there is no elution overlap between i) the flagged peptides from different fractions within one pool or between pools and ii) the flagged peptides and the unaltered peptides.