Compounds and methods for double labelling of polypeptides to allow multiplexing in mass spectrometric analysis

A marker, labeling technology, applied in the field of compounds and methods for double-labeling of peptides to allow multi-channelization in mass spectrometry analysis, can solve the problems of lack of automation and reproducibility, time-consuming analysis, etc., to achieve multi-channel Improved performance and reduced analysis time

Inactive Publication Date: 2009-09-23
KONINKLIJKE PHILIPS ELECTRONICS NV
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AI Technical Summary

Problems solved by technology

2D-GE is excellent in resolution (>5000 proteins), but suffers from lack of automation and reproducibility
While amenable to the analysis of multiple samples, the iTRAQ technique has the obvious disadvantage that it requires MS / MS for each MS signal (for both unlabeled and labeled peptides) for relative quantification of the reporter signal
In practice, this is not feasible when the starting material is of high complexity, such as in human serum or other bodily fluids, as the analysis would be very time-consuming

Method used

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  • Compounds and methods for double labelling of polypeptides to allow multiplexing in mass spectrometric analysis
  • Compounds and methods for double labelling of polypeptides to allow multiplexing in mass spectrometric analysis
  • Compounds and methods for double labelling of polypeptides to allow multiplexing in mass spectrometric analysis

Examples

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Embodiment 1

[0239] Example 1: Relative expression levels of individual peptides using isotopic and isobaric labeling Determination of

[0240] Using labeling reagents comprising unique combinations of isobaric and isotopic label components, the relative amounts of individual peptides in a mixture of labeled peptides can be determined by different combinations of isobaric and isotopic labels. This is exemplified by the following theoretical examples. Eight samples (1 to 8) of internal peptides were labeled according to the scheme in Table 4 with isotopic labels a or b and combinations of isobaric labels A, B, C and D.

[0241] Table 4: Determination of Relative Concentrations of Labeled Peptides in the Mixture

[0242]

[0243] Thereafter, all samples were pooled and subjected to chromatography in which labeled peptides behaved in the same manner irrespective of the specific isotopic / isobaric label component combination on the peptide. Differentially labeled versions of the peptid...

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Abstract

The present invention describes double labelling reagents with both an isotopic and isobaric label component suitable for differentially labelling different - protein samples. After labelling of the individual protein samples, all samples are pooled. Peptides from the pooled samples are isolated and analysed by mass spectrometry for determining the relative concentration of each differentially double-labelled polypeptide.

Description

technical field [0001] The present invention provides methods and tools for screening targets by mass spectrometry. More particularly, the methods and tools of the present invention allow the parallel screening of multiple samples by liquid chromatography mass spectrometry using a combination of isotopic and isobaric peptide labeling processes. Background technique [0002] The analysis and identification of proteins from highly complex mixtures requires enormous resolving power. Two methods commonly used to resolve such mixtures are 2-dimensional gel electrophoresis (2D-GE) and (2-dimensional) liquid chromatography ((2D)-LC). [0003] High resolution 2D-GE was introduced by Klose (1975) Humangenetik 26, 231-243 and O'Farrell (1975), J. Biol. Chem. 250, 4007-40021. 2D-GE is remarkably excellent in resolution (>5000 proteins), but suffers from a lack of automation and reproducibility. Furthermore, proteins such as hydrophobic membrane proteins, basic proteins, acidic pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6848
Inventor R·霍夫曼
Owner KONINKLIJKE PHILIPS ELECTRONICS NV
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