Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Multiplex analysis of stacked transgenic protein

A protein, transgenic plant technology, applied in the field of high-throughput analysis, can solve the problem of time-consuming and hinder the use of mass spectrometry

Active Publication Date: 2012-05-16
CORTEVA AGRISCIENCE LLC
View PDF5 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The gel separation step in this method is a time-consuming process that prevents the use of mass spectrometry in high-throughput applications

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
  • Multiplex analysis of stacked transgenic protein
  • Multiplex analysis of stacked transgenic protein
  • Multiplex analysis of stacked transgenic protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0093] Six different transgenic proteins (Cry1F, Cry34, Cry35, AAD-1, AAD-12 and PAT) were selected for development of LC\MS\MS multiplex analysis. Detect and identify individual proteins in a single injection of a complex protein mixture by mass spectrometry.

[0094] The first format of the multiplex analysis was to proteolytically digest the six proteins separately, then enrich the resulting protein peptides into proteolytically digested plant tissue extracts and use LC\MS\MS for analysis in a single Injection was performed to detect specific precursor / fragment ions for each of the six proteins. The methodology developed during this first multiplex format was then applied to multiplex detection of four proteins expressed in current inbred maize breeding material.

Embodiment II

[0096] LC\MS\MS multiple detection in plants

[0097] Table 1 lists the concentration of each individual stock protein received, and the resulting dilution of each protein when subjected to trypsinization. Prior to digestion with the protease trypsin, all proteins were buffer exchanged to 25 mM ammonium bicarbonate, pH 7.9 (SIGMA) to ensure efficient digestion conditions. Aliquots from each stock protein (see Table 1) were transferred to sterile 1.5 mL eppendorf tubes and made up to 100 μL using 25 mM ammonium bicarbonate, pH 7.9. Zeba Desalt Spin Column (Pierce #89882) was used for buffer exchange of each protein according to the manufacturer's recommendations. Three spin washes of the Zeba column were performed, each wash using 300 μL of 25 mM ammonium bicarbonate, pH 7.9, and spinning at 1500 g for 1 min. A 100 μL aliquot of each sample protein was then applied to the surface of the Zeba column resin and spun at 1500g for 2 minutes. The buffer-exchanged material was then...

Embodiment III

[0109] A single LC\MS\MS multiplex targeted MRM analysis for all six DAS proteins was then constructed using the precursor / fragment ion data generated from the individual protein analyses. The peptides were first subjected to LC\MS\MS multiplex detection by fortifying ammonium bicarbonate buffer (25 mM, pH 7.9) with tryptic digested material from each of the six proteins. Approximately 5-20 fmol of each protein was injected onto the column for the initial multiplex-6 analysis of the boosted buffer.

[0110] Figure 1-6 Extracted ion chromatograms for each of the six proteins detected in a single injection are shown. in particular, figure 1 LC\MS\MS multiplex detection of AAD-1 is shown. The data are LC\MS\MS ion chromatograms for the extraction of AAD-1 ((R)-2,4-dichlorophenoxypropionate dioxygenase), which contains AAD in the corn seed extract -12, Cry1F, Cry34, Cry35 and PAT were detected in a single injection.

[0111] figure 2 LC\MS\MS multiplex detection of AAD-12 ...

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 relates to methods for multiplex analysis of complex protein samples from plants using mass spectroscopy. In some embodiments, the disclosure concerns methods for maintaining a transgenic plant variety, for example by analyzing generations of a transgenic plant variety for presence and concentration of multiplexed transgenic proteins.

Description

[0001] This application claims priority to Provisional Application Serial No. 61 / 183,777, filed June 3, 2009 in the United States Patent and Trademark Office, which is hereby incorporated by reference. technical field [0002] The present invention generally relates to high throughput analysis of plant traits. In certain embodiments, the present invention relates to methods of high throughput analysis and determination of plant protein quantities by mass spectrometry using a single injection of complex protein samples derived from plants and plant tissues. Certain embodiments further relate to methods of genotyping transgenic plants and maintaining expression in those plants having desired plant traits. Background technique [0003] The increasing use of recombinant DNA technology to generate transgenic plants for commercial and industrial use requires the development of high-throughput methods for the analysis of transgenic plant lines. Such methods are needed to maintain ...

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): B01D59/44C12P21/06C07K1/36
CPCG01N33/6848G01N2333/415
Inventor J.劳里J.弗卢克
Owner CORTEVA AGRISCIENCE LLC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com