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

a proteome and qualitative and quantitative technology, applied in biochemistry apparatus and processes, peptide preparation methods, component separation, etc., can solve the problems of difficult analysis using this method, high labor intensity, and sequential 2d-pages, and achieve the effects of high acidity or basic protein, high labor intensity, and high labor intensity

Inactive Publication Date: 2005-09-08
PRONOTA NV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0002] 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.

Problems solved by technology

However, 2D-PAGE is sequential, labour intensive, and difficult to automate.
In addition, specific classes of proteins, such as membrane proteins, very large and small proteins, and highly acidic or basic proteins, are difficult to analyze using this method.
Another significant flaw lies in its bias toward highly abundant proteins, as lower abundant regulatory proteins (such as transcription factors and protein kinases) are rarely detected when total cell lysates are analyzed
In most complex biological samples, the proteolysis of the proteins will produce thousands of peptides and this overwhelms the resolution capacity of any known chromatographic system.
It results in the co-elution and therefore inefficient separation and isolation of individual peptides.
In addition, the resolving power of mass spectrometry coupled with such chromatography is not sufficient to adequately determine the mass of the individual peptides.
While this strategy improves the separation of the complex mixture in its individual components, the resolving power of this approach is still largely insufficient to reproducibly identify the constituting peptides in biological samples.
Further disadvantages of the DALPC method are the incompatibility with the analysis of low-abundance proteins and the fact that the method cannot be used quantitatively.
However, disadvantages are that an affinity purification step generally necessitates the use of greater amounts of starting material because of the loss of material during the purification step.
The method also fails for proteins that contain no cysteine residues.
This means that there is no optimal chromatographic separation and a less efficient mass spectrometric detection of the modified peptides.

Method used

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

Examples

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example 1

Specific Chemical Alteration of Methionine-Residues

[0190] A protein peptide mixture was generated according to the method described in the invention and the relatively rare amino acid methionine was selected for alteration. As documented in the literature, one approach to alter methionine is by chemical oxidation, which can lead to sulfoxide-formation and to sulfone-formation. Peptides comprising methionine can be converted into their sulfone derivatives by using strong oxidizing conditions such as with performic acid or other per-acids (Toennies and Homiller, 1942 and Hirs, 1956). It is known that the stronger oxidizing conditions are rather harsh and not selective enough. The formation of methionine-sulfoxide proceeds upon contact of methionine with the air. However, in the presence of 0.5% H2O2 at room temperature and low pH (1% TFA), this reaction is completed in less than 30 minutes. Interestingly, under these mild conditions, it was observed that both cysteine and tryptophan,...

example 2

Specific Chemical Alteration of Cysteine-Residues

[0192] A protein peptide mixture is generated with one of the methods described herein before and a specific chemical alteration of cysteine residues is carried out. Said alteration is for instance based on the specific conversion of cysteine peptides into a more hydrophilic derivative, which undergoes a hydrophilic shift during reversed phase HPLC. Several reagents can fulfill these requirements. For instance, reactions with iodoacetamide, iodoacetate, ethyleneimine, bromoethylamine, acrylamide and 4-vinyl pyridine, all convert cysteine into compounds that behave more hydrophilic in reverse phase-conditions. In addition these compounds all undergo oxidation by H2O2 resulting in the formation of their corresponding sulfoxide derivatives, which are even more hydrophilic. It is important to mention that the shift due to oxidation is less pronounced here than in the case of the methionine oxidation. However, when combining the shifts be...

example 3

Specific Chemical Alteration of the Sum of Methionine and Cysteine Residues

[0197] The procedure is identical to the procedure for specifically altering cysteine-residues (see Example 2) with the exception that the pre-oxidation step of the methionine residues is omitted. The reaction sequence starts with the reduction of a protein mixture with tributyl phosphine, followed by enzymatic or chemical cleavage. The protein peptide mixture is separated by RP-HPLC and each fraction is altered by reaction with acrylamide, immediately followed by oxidation with H2O2. The methionine-peptides are now oxidized together with the altered cysteine peptides and both types of flagged peptides show a hydrophilic shift when chromatographically separated using similar conditions as in the primary run. FIG. 5 summarizes the various reaction sequences in the Met-sorting, Cys-sorting and Met+Cys-sorting modes.

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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.

Description

RELATED APPLICATIONS [0001] The present application claims priority from U.S. provisional patent application Ser. No. 60 / 323999, filed on Sep. 20, 2001, and from U.S. provisional patent application Ser. No. 60 / 318749, filed on Sep. 12, 2001. The present application also claims priority from U.S. provisional patent application Ser. No. 60 / 278171, filed on Mar. 22, 2001. The present application additionally claims priority from PCT application no. PCT / EP02 / 03368, filed Mar. 22, 2002. All of the above applications are expressly incorporated by reference.SUMMARY OF THE INVENTION [0002] 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 t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K1/16C07K1/20G01N27/62C07K1/36C12P21/02G01N30/02G01N30/06G01N30/34G01N30/46G01N30/72G01N30/80G01N30/84G01N30/88G01N33/48G01N33/50G01N33/68
CPCB01D15/1871C07K1/36G01N30/461G01N30/467G01N30/468Y10S435/803G01N30/80G01N33/6842G01N33/6848G01N2030/8411G01N30/7233
Inventor VANDEKERCKHOVE, JOELGEVAERT, KRIS
Owner PRONOTA NV
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