Polypeptide marker for early diagnosis of diabetes mellitus

A diabetes diagnosis and marker technology, applied in the biological field, can solve the problems of changing protein molecular quality and detection difficulties

Inactive Publication Date: 2014-07-02
BEIJING INSTITUTE OF TECHNOLOGYGY
View PDF0 Cites 18 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Each post-translational modification may alter the molecular mass of the protein, making detection difficult for mass spectrometry, an instrument based on molecular weight determination

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Polypeptide marker for early diagnosis of diabetes mellitus
  • Polypeptide marker for early diagnosis of diabetes mellitus
  • Polypeptide marker for early diagnosis of diabetes mellitus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 In vitro establishment of a simulated in vivo glycosylation model based on HAS single protein screening of glucose-sensitive peptides

[0084] 1. Preparation of in vitro glycosylation samples

[0085] The preparation of in vitro glycosylation samples can basically be based on the methods reported in the literature [Sattarahmady, N., et al., Formation of the molten globule-like state during prolonged glycation of human serum albumin. [J]. Biochim Biophys Acta, 2007.1770( 6): p.933-42]. Unmodified human serum albumin (HSA) (purity ≥ 96%, purchased from Beijing Baierdi Biotechnology Co., Ltd.) was prepared in 50mM PBS (pH7.4) buffer solution with a physiological concentration (40mM / mL ), were incubated with four different concentrations of D-glucose in a 37°C water bath for 10 days, 20 days, and 30 days, so that the molar ratios of HSA to D-glucose were 1:10, 1:41.5, 1:83, and 1:415. The 4 glucose concentrations are based on physiological glucose concentration ...

Embodiment 2

[0174] Example 2 Validation of 11 target peptides of the present invention in healthy human plasma samples

[0175] 1. Source of plasma samples and determination of protein concentration

[0176] Plasma samples from 12 healthy people (6 males and 6 males) were provided by Beijing Institute of Technology Hospital, and all biochemical indicators of the plasma donors were normal after testing.

[0177] Mix equal volumes of 12 samples, and vortex to make the mixture even. The protein concentration in the mixed sample was determined by Coomassie brilliant blue method (Bradford) to be 82.4 μg / μL.

[0178] 2. Preparation of mixed plasma in vitro glycosylation samples

[0179] The preparation of the in vitro glycosylation sample of mixed plasma is similar to the preparation of the standard protein in vitro glycosylation sample (see item 1 of Example 1 for the method). 12 cases of mixed plasma were incubated with 4 different concentrations of D-glucose in a 37°C water bath for 10 days...

Embodiment 3

[0249] Example 3 Verification and Application of Polypeptide Markers for Diabetes Detection in Clinical Samples

[0250] Through Example 2, the present invention has screened 11 target peptides through the standard HSA in vitro glycosylation model. Then, the above target peptides were initially verified by using healthy human plasma samples under the same in vitro model conditions, and 5 peptides that could not be detected in complex plasma samples were eliminated. Next, a large number of actual clinical samples from healthy people, type 2 diabetes patients and people with impaired glucose tolerance need to be used to further verify the six screened peptide biomarkers that may become diabetes. The experimental process is as follows Figure 24 shown.

[0251] It can be seen from the experimental flow chart that two main experimental steps are enzymatic hydrolysis and HPLC-MS detection from the sample collection to the detection to obtain the peak area ratio of the target pept...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
strengthaaaaaaaaaa
Login to view more

Abstract

The invention provides a polypeptide marker for early diagnosis of diabetes mellitus. The operating method comprises the following steps: establishing an in-vitro simulated glycosylation model by selecting human peripheral blood high-abundant protein HAS, performing enzyme digestion under optimized conditions, performing 18O marking on a blank control group sample subjected to enzyme digestion, mixing the blank control group sample with a reaction group sample subjected to parallel treatment by using 16O according to a volume percent of 1:1, and performing HPLC/ESI-TOFMS detection. According to quantitative analysis of HSA peptide fragments, a peptide fragment, which is the most difficultly modified by glucose and is shown as SEQ ID NO.1, serves as an internal standard peptide fragment, a ratio of a peptide fragment which is easily subjected to glycosylation modification to the peak area of the internal standard peptide fragment is used for measuring the glycosylation modification degree of a glucose sensitive peptide, and according to a proteomics method, three peptide fragments, namely FKDLGEENFK, LDELRDEGK and KVPQVSTPTLVEVSR are finally discovered to serve as biological markers to be used for early diagnosis of diabetes mellitus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a polypeptide marker for detecting early diabetes and its application. Background technique [0002] Diabetes mellitus is a group of metabolic diseases characterized by chronic increase in blood glucose (referred to as blood sugar) level, which is caused by defects in insulin secretion and (or) action. At present, the level of fasting blood glucose and glycosylated hemoglobin is used as the direct standard for clinical diagnosis of diabetes in medicine. However, for people with pre-diabetes, the blood sugar level will not obviously be the first to show a high state, but due to the decline in the body's ability to regulate blood sugar levels, it is manifested as fluctuating high and low blood sugar levels. The determination of glycosylated hemoglobin usually reflects a person's relatively long-term (8–12 weeks) blood sugar status. Usually, once the levels of fasting blood glucose an...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K7/06G01N30/02G01N33/68
Inventor 邓玉林张玫
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap