Functional reagent containing trimethylpiperidine group and triphenylphosphine group as well as preparation method and application of functional reagent

A functionalization and phenyl technology, applied in the field of analytical chemistry, can solve the problems of affecting the stability of protein post-translational modification, difficult elution of modified peptides, unfavorable identification, etc., to increase ionization efficiency, improve detection, and increase identification sensitivity and the effect of data size

Active Publication Date: 2020-02-07
BEIJING PROTEOME RES CENT +1
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, this strategy cannot avoid non-specific interference caused by the binding of streptavidin magnetic beads to endogenous biotinylated proteins in cells
Moreover, due to the extremely high affinity between biotin and streptavidin, it is difficult for the enriched post-translationally modified peptides to be eluted from the magnetic beads, making it impossible to perform mass spectrometry analysis and site identification
Peptide elution using strong elution conditions or chemical fragmentation methods will affect the stability of protein post-translational modifications, which is not conducive to their identification

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
  • Functional reagent containing trimethylpiperidine group and triphenylphosphine group as well as preparation method and application of functional reagent
  • Functional reagent containing trimethylpiperidine group and triphenylphosphine group as well as preparation method and application of functional reagent
  • Functional reagent containing trimethylpiperidine group and triphenylphosphine group as well as preparation method and application of functional reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, TFT reagent is synthesized as figure 2 As shown, the specific steps are as follows:

[0063] 1.1 Bromoacetic acid substitution reaction to obtain compound 2: Dissolve bromoacetic acid (2.0g, 14.4mmol) in pure water (8mL), add 3.3M NaOH (5mL), adjust the pH to 14, cool to 0°C, and add compound 1 dropwise (1 mL, 11.9 mmol), react in an ice bath for 1 hour, then slowly rise to room temperature for 3 days. The reaction was detected by thin-layer chromatography, extracted three times with dichloromethane, and the product was washed with brine and dried over anhydrous sodium sulfate. It was further purified by silica gel column chromatography (dichloromethane / methanol=20 / 1) to obtain compound 2 (1.3 g, 7.7 mmol, 65%).

[0064] 1.2 Amidation reaction of compound 3 and compound 2 and catalytic reduction reaction of product 4: Dissolve compound 2 (3.0 g, 17.5 mmol) in dry dichloromethane (13 mL), slowly add thionyl chloride dropwise under ice bath (7.77mL, 95.8...

example 3

[0069] Example 3, novel TFT reagent to standard N 3-Evaluation of derivatization efficiency, mass spectrometry detection sensitivity, and MS / MS fragmentation behavior of O-GlcNAc glycopeptides

[0070] Synthetic four criteria N 3 -O-GlcNAc glycopeptides are LNPAVT[N 3 -O-GlcNAc]CAGK (peptide 1), RQLFVT[N 3 -O-GlcNAc]VVK (peptide 2), AQPVQS[N 3 -O-GlcNAc]KPQK (peptide 3) and AAAPAPVS[N 3 -O-GlcNAc]EAVCR (peptide 4), dissolve 10μg glycopeptide (1μg / μL) in PBS, add TFT reagent with a final concentration of 0.5mM, mix evenly with a vortex instrument, seal with a parafilm, and put React in a constant temperature incubator at 37°C for 4 hours. The labeled samples were desalted with a C18 column, freeze-dried, and detected by liquid chromatography-mass spectrometry.

[0071] The labeled peptide was detected by MALDI-TOF-MS, and there was no residual unlabeled standard azide glycopeptide signal in the mass spectrum, indicating that the TFT labeling efficiency reached 100%. Then...

example 4

[0072] Example 4, the antibody resin functionalized by TFT reagents to the N of doped BSA enzyme-cleaved peptides 3 Enrichment efficiency of -O-GlcNAc standard glycopeptides

[0073] In order to evaluate the feasibility of the enrichment method described in the present invention, we first analyzed the N 3 -O-GlcNAc standard glycopeptides were enriched and identified:

[0074] 1) Preparation of TFT functionalized antibody: TFT reagent was added to anti-TMT antibody resin (purchased from thermo fisher scientific company) containing 50 nmol / mL, and incubated at room temperature for 1 h.

[0075] 2) Enrichment: N 3 -O-GlcNAc standard glycopeptides were mixed with BSA enzyme-cleaved peptides at a mass ratio of 1:100, mixed with 25 μL of the TFT functionalized antibody resin obtained in 1), and incubated at room temperature for 4 h. After enrichment, use 100 μl IP buffer containing 0.05% Tween-20 (100 mM HEPES, 250 mM NaCl, 0.2 mM NaCl 2 HPO 4 , pH=7.4) and washed 3 times to re...

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

No PUM Login to view more

Abstract

The invention provides a compound provided with a trimethylpiperidine group at one end and a triphenylphosphine group at the other end, and a structural formula of the compound is shown as a followingformula I. The reagent is applied to one-step derivation, enrichment and mass spectrum identification of an azide-labeled posttranslational modification peptide fragment. The derivation of a TFT reagent can effectively enhance the hydrophobicity and alkalinity of the posttranslational modification peptide fragment, so that the ionization and fragmentation efficiencies of the peptide fragment in amass spectrum are increased, and the sensitivity of the mass spectrum identification and spectrogram analysis are remarkably improved. In addition, the TFT reagent can be specifically and reversiblycombined on antibody resin due to the moderate affinity (kD=86 pM) of the trimethylpiperidine group of the TFT reagent and an anti-TMT antibody, so that the selective enrichment, simple release and efficient recovery of the azide-labeled posttranslational modification peptide fragment with low abundance are realized. By utilizing the strategy, the high-selectivity enrichment and deep-coverage identification (more than 3800 high-assurance O-GlcNAc glycopeptides) of azide-labeled O-GlcNAc glycopeptides in complex samples of standard N3-O-GlcNAc glycopeptides and Hela cells are successfully realized.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and specifically relates to a chemical derivatization-enrichment reagent (TFT reagent for short) with a trimethylpiperidine group at one end and a triphenylphosphine group at the other end, and a preparation method and application thereof. The reagent is achieved through the "Staudinger connection" reaction of triphenylphosphine and azide-labeled protein post-translational modification, and the specific and reversible binding of trimethylpiperidine group to Anti-TMT antibody coupling resin. One-step derivatization, enrichment, and mass spectrometric identification of post-translationally modified proteins / peptides. Background technique [0002] Protein post-translational modification plays a very important role in many cellular processes and regulates various functions of proteins, such as: protein synthesis, biological activity, cellular localization, protein-protein interaction, etc. Scaled...

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): C07F9/59C07K5/023G01N27/62G01N1/40
CPCC07K5/0202G01N27/62G01N1/40
Inventor 秦伟捷钱小红霍变变张万军
Owner BEIJING PROTEOME RES CENT
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