Molecular weight correction standard substance kit for polypeptide or protein spectrum detection as well as preparation method and use method of molecular weight correction standard substance kit

A molecular weight internal standard and standard technology, applied in the field of mass spectrometry, can solve the problems of high purchase cost, no disclosure of characteristic peaks of peptide standards, and inability to take into account molecular weight calibration and optimization. cost effect

Pending Publication Date: 2020-06-05
HANGZHOU WELL HEALTHCARE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still belongs to external standard calibration in operation, and does not disclose the characteristic peaks of peptide standards extracted from biological samples, and there is still the problem of parallelism comparison of test results in different laboratories
[0005] At present, the purchase cost of commercial peptide standard products on the market is relatively high, and the molecular weight range is about 2000-20000Da, which cannot take into account the molecular weight calibration and optimization of macromolecules below 1000Da and above 10000Da. Broad biological sample peptide or protein spectrum detection
Both the external standard method and the internal standard method of peptide molecular weight calibration have their own disadvantages

Method used

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  • Molecular weight correction standard substance kit for polypeptide or protein spectrum detection as well as preparation method and use method of molecular weight correction standard substance kit
  • Molecular weight correction standard substance kit for polypeptide or protein spectrum detection as well as preparation method and use method of molecular weight correction standard substance kit
  • Molecular weight correction standard substance kit for polypeptide or protein spectrum detection as well as preparation method and use method of molecular weight correction standard substance kit

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Experimental program
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preparation example Construction

[0054] A method for preparing a molecular weight calibration standard kit for polypeptide or protein spectrum detection, comprising the following steps:

[0055] Step 1, preparation of peptide or protein dry powder standard:

[0056] 1) Mix multiple samples in equal volumes to prepare a large number of standard samples;

[0057] 2) using nanomaterials to enrich the polypeptides and proteins in the standard sample, and eluting with the eluent; as an example, the eluent is 30-60% CAN and 0.5-5% TFA;

[0058] 3) Freeze and vacuum-dry the peptide eluate to obtain a peptide or protein dry powder standard;

[0059] Step 2, Determination of the molecular weight of the peptide in the peptide or protein dry powder standard:

[0060] 1) Dissolving the peptide or protein dry powder standard with the dry powder solution to obtain a peptide standard solution, adding a commercial standard to the peptide standard solution as an internal standard to prepare a peptide standard solution conta...

Embodiment 1

[0087] Kit, peptide or protein dry powder standard, dry powder solution, matrix solution, matrix, molecular weight external standard calibration list, molecular weight internal standard calibration list; the ratio of peptide or protein dry powder standard to matrix is ​​3:10;

[0088] 1. Preparation of mass spectrometer molecular weight calibration standards for human serum peptide or protein spectrum detection:

[0089] 1) Select 50 cases of healthy physical examination serum samples according to age and gender, take 800uL of each sample, mix them evenly, and divide them into centrifuge tubes according to 120uL / tube, store them at -80°C, and use them as standard serum samples;

[0090] 2) Mix standard serum samples, ultrapure water, and mesoporous materials (the mixing ratio is V:V:M=100-200uL:900-800uL:0.5-1mg), and perform steps such as incubation, centrifugation, and washing;

[0091] 3) Add 1-1.2 mL of eluent of 30-60% CAN and 0.5-5% TFA to the mesoporous material enriche...

Embodiment 2

[0107] Embodiment 2: Only the formula of kit is different from embodiment 1;

[0108] Kit, including: peptide or protein dry powder standard, dry powder solution, matrix solution, matrix, molecular weight external standard calibration list, molecular weight internal standard calibration list; the ratio of peptide or protein dry powder standard to matrix is ​​1:10;

[0109] 1. Preparation of mass spectrometer molecular weight calibration standards for human serum peptide or protein spectrum detection:

[0110] 1) Select 50 cases of healthy physical examination serum samples according to age and gender, take 800uL of each sample, mix them evenly, and divide them into centrifuge tubes according to 120uL / tube, store them at -80°C, and use them as standard serum samples;

[0111] 2) Mix standard serum samples, ultrapure water, and mesoporous materials (the mixing ratio is V:V:M=100-200uL:900-800uL:0.5-1mg), and perform steps such as incubation, centrifugation, and washing;

[0112...

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Abstract

The invention discloses a molecular weight correction standard substance kit for polypeptide or protein spectrum detection as well as a preparation method and a use method of the molecular weight correction standard substance kit. The kit comprises a polypeptide or protein dry powder standard substance, a dry powder dissolving solution, a matrix dissolving solution, a matrix, a molecular weight external standard correction list and a molecular weight internal standard correction list, the ratio of the polypeptide or protein dry powder standard substance to the matrix is as follows: 1-6 parts of polypeptide or protein dry powder corresponds to 10 parts of matrixes; the preparation method of the kit not only is low in cost, but also can selectively prepare a calibration product according toa specific calibration purpose, and molecular weight correction considers both small molecule and large molecule ranges; the molecular weight correction method provided by the invention has the advantages that the operation convenience of an external standard method and the high precision of an internal standard method are realized, and meanwhile, the potential influence of the addition of an exogenous internal standard substance in a sample on the polypeptide fingerprint spectrum can also be avoided; the kit can be applied to application scenarios such as liquid biopsy and microbial identification based on a mass spectrometry technology.

Description

technical field [0001] The invention relates to the technical field of mass spectrometry, in particular to a molecular weight calibration standard kit for polypeptide or protein spectrum detection and its preparation method and use method. Background technique [0002] Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is a soft ionization mass spectrometry technique suitable for the analysis of biological macromolecules and mixture samples. Compared with other analysis methods, MALDI-MS has the advantages of high sensitivity and high throughput, but the analysis results are greatly affected by system errors and fluctuations in test conditions. In order to improve the accuracy of the analysis results, it is generally necessary to calibrate the molecular weight of the instrument through a standard. The calibration methods are divided into external standard method and internal standard method. The external standard method refers to firstly using the mole...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/96G01N27/62
CPCG01N33/96G01N33/6848
Inventor 余捷凯陈锡胜栾春燕邬建敏
Owner HANGZHOU WELL HEALTHCARE TECH CO LTD
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