Polypeptide deriving method and application thereof to MALDI-TOF-MS detection for small molecule compound

A technology of MALDI-TOF-MS and small molecule compounds, which is applied in the field of mass spectrometry detection, can solve the problems of cumbersome preparation of derivatization reagents, unsuitable detection of small molecular substances of metabolites, matrix interference of target analytes, etc., to facilitate quantitative analysis, High universality and easy operation

Inactive Publication Date: 2016-06-22
WUHAN UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, although MALDI-TOF-MS has significant advantages in the detection of macromolecular substances, it is not suitable for the detection of small molecular substances such as metabolites, mainly because of the commonly used matrix CHCA, DHB, 3-HPA, 2,4 , 6-THP, etc. in the small molecule (<500Da) segment will have serious matrix interference to the target analyte
In order to overcome this shortcoming, the methods reported in the existing literature are mainly divided into two categories: 1) the matrix with little matrix interference is adopted, such as porous silicon, nano-microspheres, metal oxides, carbon materials, etc., but there is still no convenient, Widely applicable matrices have been developed; 2) Design macromolecular derivatization reagents to derivatize small molecule analytes. The preparation of derivatization reagents reported in the existing literature is usually very cumbersome and has certain limitations

Method used

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  • Polypeptide deriving method and application thereof to MALDI-TOF-MS detection for small molecule compound
  • Polypeptide deriving method and application thereof to MALDI-TOF-MS detection for small molecule compound
  • Polypeptide deriving method and application thereof to MALDI-TOF-MS detection for small molecule compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] [Example 1] Comparison of MALDI-TOF-MS detection sensitivity before and after derivatization of several aldehyde compounds and sulfhydryl compounds

[0038] Table 1 Comparison of MALDI-TOF-MS detection sensitivity before and after polypeptide derivatization.

[0039]

[0040]

[0041] ND: 1 nmol not detected.

[0042] Analyte unpeptidated derivatization sensitivity: Mix 1 μL test solution with 1 μL matrix (CHCA) to spot the plate; after drying at room temperature, enter matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, and measure its LOD.

[0043] Analyte peptide derivatization sensitivity: after derivatization of small molecule analytes, mix 1 μL of test solution with 1 μL matrix (CHCA) to spot the plate; after drying at room temperature, enter matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to measure its LOD .

Embodiment 2

[0044] [Example 2] Determination of detection limit after derivatization of 3-pyridine formaldehyde and mercaptoethanol

[0045] Different amounts of small molecule analytes (3-pyridinecarbaldehyde, mercaptoethanol) were subjected to peptide derivatization using the above method, mixed with 1 μL of the test solution and 1 μL of matrix (CHCA) to spot the plate; after drying at room temperature, enter the matrix-assisted laser desorption ionization - Time-of-flight mass spectrometry analysis to obtain detection limits for small molecule compounds.

Embodiment 3

[0046] [Example 3] Analysis and detection of sulfhydryl compounds in the urine of lung cancer patients

[0047] Urine samples from lung cancer patients were obtained from Hubei Cancer Hospital through standard clinical channels. The collected urine samples were stored in a -80°C refrigerator. Before sample analysis, the urine sample was centrifuged at 12,000 r / min for 5 min at 4°C, and the supernatant was taken for testing. For the analysis of sulfhydryl compounds, 100 μL of urine samples were reacted with 100 nmol of TCEP at 45 °C for 60 min to reduce disulfide bonds. Then, 20μL urine sample was mixed with 20nmol polypeptide P7 or 7 P7 was added to a phosphate buffer solution with a pH of 6, derivatized at 4°C for 0.5h, 1 μL of the reaction solution was mixed with 1 μL of matrix (CHCA) and spotted on the plate; after air-drying at room temperature, it entered matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, using positive ion reflectance mode a...

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Abstract

The invention discloses an analysis method for combining chemical derivation with matrix auxiliary laser desorption ionization-time of flight mass-spectrometric technique (MALDI-TOF-MS). According to the method provided by the invention, polypeptide is adopted for deriving aldehyde and sulfydryl micromolecule metabolites, and after the to-be-detected micromolecule matter is derived, the MALDI-TOF-MS detection analysis is performed. After the micromolecule matter is subjected to polypeptide treatment, the simple single quasi-molecular ion peak [M+H]+ is acquired, so that the distinguishing degree of the micromolecule matter in a mass spectrum graph can be obviously improved and the detection sensitivity of the micromolecule matter in the mass spectrum is greatly increased. The method has the advantages of high sensitivity, strong universality, simple and convenient operation, no matrix interference and less sample loss; the sulfhydryl compound in ultralow content in a body fluid can be quantitatively detected, without preparation for a complex matrix and a deriving reagent; and the method has a huge application prospect in search on metabonomics.

Description

technical field [0001] The invention belongs to the technical field of mass spectrometry detection, and in particular relates to a method of polypeptide derivatization combined with MALDI-TOF-MS and its application in the field of metabolomics. Background technique [0002] Metabolomics is another research field that characterizes the status of living systems after genomics and proteomics; the detection of metabolites in organisms has profound significance for the study of gene expression, exploration of life processes, disease diagnosis and prevention, etc. Therefore, the analysis of metabolites is an important research direction in the field of analytical chemistry. [0003] As an important means of metabolomics research, mass spectrometry has significant advantages such as high sensitivity, good specificity, and strong qualitative ability. Currently, the most commonly used mass spectrometric detection technique for small molecule metabolites is liquid chromatography-mass...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/64
CPCG01N27/64
Inventor 冯钰锜袁必锋唐峰
Owner WUHAN UNIV
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