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Use of magnetic bead supported matrix and MS for judging mass spectrometry polypeptide spectrum and protein fingerprint

A protein and magnetic bead technology, applied in the field of protein fingerprinting and protein fingerprinting of biological sample analysis, can solve problems such as unseen, limited gel capacity and sensitivity, and inability to observe molecular weight.

Inactive Publication Date: 2008-04-30
许洋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, the method provides only two characteristics of biomolecules - mass and isoelectric point (pI)
Second, the resolution in each dimension is limited by the resolving power of the gel
For example, it is often difficult to distinguish between biomolecules that differ by less than 5% in mass or that differ in pI
Third, gels have limited capacity and sensitivity and may not be able to detect small amounts of expressed biomolecules
Fourth, small proteins or peptides with a molecular weight below about 10-20 kDa cannot be observed
However, there are currently only 289 protein molecular weights in human blood that can be used clinically, and there is no literature that uses the matrix method supported by magnetic beads to identify and analyze protein fingerprints and interpret tens of thousands of molecular weight bands (Anderson NL and Anderson NG , Molecular & Celular Proteomics 1:845-867; 2002)

Method used

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  • Use of magnetic bead supported matrix and MS for judging mass spectrometry polypeptide spectrum and protein fingerprint
  • Use of magnetic bead supported matrix and MS for judging mass spectrometry polypeptide spectrum and protein fingerprint
  • Use of magnetic bead supported matrix and MS for judging mass spectrometry polypeptide spectrum and protein fingerprint

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Significance of Differential Expression of Protein Fingerprints in Biological Samples

[0066] (1) Experimental method

[0067] 1. Materials

[0068] 1. Specimen sources: Serum from 10 normal people and 8 patients with pneumonia.

[0069] 2. Reagents: Protein A and G, human standard serum, human growth hormone, insulin (molecular weight 5807Da), anti-insulin antibody, anti-human serum amyloid A antibody, anti-fibrinopeptide A antibody; acetonitrile, trifluoroacetic acid, SINAPINIC Acid (Sinapinic acid), CHAPS, Urea, NaAC, Tris-HCl were purchased from Sigma or donated.

[0070] 3. Quality control: add human growth hormone (Sigma) to human standard serum (Sigma), and adjust the concentration of human growth hormone in standard human serum to be 50 ng / ml.

[0071] 2. Method

[0072] 1. Collection of samples: After the whole blood is collected, draw the serum and store it at -80°C; take out the serum sample in the refrigerator at -80°C, and put it on the ice b...

Embodiment 2

[0081] Example 2 Sequence identification of serum biomarkers

[0082] Because the biomarkers in this invention are identified by mass spectrometry and magnetic bead support matrix and antibody column, they can be detected by mass spectrometry to directly know their specific identity. This method is more accurate than antibody-based ELISA and immunofluorescence methods.

[0083] The serum 1536.7Da biomarker protein was reduced from a fibrinopeptide A antibody column using MALDI-Qq-TOF mass spectrometry (MS / MS), post-source fragmentation (PSD) and protein ladder sequencing Sort. By breaking molecules into pieces, protein ladders can be generated. This gradient is then analyzed by mass spectrometry. The biomarker and its chemical structure were identified as fibrinopeptide A (arranged from N-terminus to C-terminus):

[0084] N-terminal Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Gly-Val-Arg C-terminal

[0085] Check the chemical structure of the normal fibrinopep...

Embodiment 3

[0088] Example 3 Normal Human Serum Protein Fingerprint and Mass Spectrometry Peptide Spectrum

[0089] Quantitative mass spectrometry regulation: Before each test, use the standardized quality control serum of mass spectrometry to adjust the standard peak 6634.0Da equal intensity for quantification in the standardized quality control serum to the maximum value of 40-50% signal intensity ( image 3 ). image 3 The fingerprints and mass spectrometry peptide spectra of two serum proteins of normal people are shown. 2746±1Da, 5909±1Da, 6634±1Da peaks can be used as standard peaks for molecular weight quality control.

[0090] in conclusion

[0091] Protein fingerprinting of biological samples captured by magnetic bead-supported matrix for mass spectrometry analysis - in a computer-readable barcode format (protein fingerprint). The present invention provides for determining the presence of a difference between a first and a second sample of a biological sample. Including A) dete...

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Abstract

The invention relates to a proteome acquiring method which utilizes the method of bead supporting stroma to capture the protein group in the biology samples, and utilizes the magnetic separator to separate the bead and the sample without eccentric samples, and then utilizes the matter hidden method to analyze. The type of the protein molecular weight obtained by the analysis is a bar code format (protein fingerprint) which can be read by a computer. The invention provides a meaning which confirms the matter hidden bar code format in a certain sample. The confirmations of the bar code format (protein fingerprint) and the meaning of the bar code belt (molecular weight) adopt the addition and subtraction, namely adding or subtracting a certain matter to confirm the meanings of the mass spectrum molecular weight or the biological sign chart. The method of the invention can be used for the identification of the protein fingerprint of the normal people and patients. The significant meaning of the protein fingerprint altas is that the protein fingerprint altas regularly inspects the dynamic changes of mass spectrum polypeptide chart, protein fingerprint and significant biology sing chart in the human activity or blood. The protein single protein fingerprint and the molecule sequence in the protein fingerprint can be used in the testing method of the reagent box of the body liquid part away from the body. The method is accurate, convenient and quick.

Description

technical field [0001] The invention relates to a novel method for analyzing proteins in biological samples, in particular to a protein fingerprint method for analyzing biological samples. The present invention also relates to protein fingerprinting in which proteomes are captured using a magnetic bead supported matrix and analyzed using a computer readable barcode format (protein fingerprinting). In the barcode format (protein fingerprint), the meaning of the barcode band (molecular weight) is determined using an addition and subtraction method, that is, adding or subtracting a certain substance to determine the meaning of its mass spectrum molecular weight. The protein fingerprinting method determines the protein fingerprint difference between the first and second (addition or subtraction of certain substances) samples of a biological sample: the difference barcode bands of the two protein fingerprints affirm the clinical significance of its molecular weight in biological sa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N30/02G01N30/86G01N33/543G01N33/577
Inventor 许洋
Owner 许洋
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