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

Substrates and internal standards for mass spectroscopy detection

一种底物、质谱分析的技术，应用在分析试剂和基于质谱法领域，能够解决苛刻分析成分、分析方法不容易适用于临床情况、麻烦等问题，达到时间少的效果

Inactive Publication Date: 2009-05-06
PERKINELMER HEALTH SCIENCES INC
View PDF0 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although lysosomal enzyme activity can be quantified using tandem mass spectrometry (Gelb MH et al., Clinical Chemistry 50: 10, 1785-1796, 2004), published analytical methods are not easily applicable to clinical situations due to cumbersome procedures and harsh Analytical components such as chloroform

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
  • Substrates and internal standards for mass spectroscopy detection
  • Substrates and internal standards for mass spectroscopy detection
  • Substrates and internal standards for mass spectroscopy detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] For each sample, 3 mm diameter discs were punched from the dried blood area on the filter paper into the wells of microcentrifuge tubes or 96-well microtiter plates. Then, the blood disk was directly incubated with an assay solution containing a substrate for Fabry disease at a final concentration of 3 mmol / L and an internal standard at a final concentration of 0.05 mmol / L. To this analytical solution, a sodium acetate buffer at a final concentration of 0.5 mol / L was also added. The assay mixture containing the blood disk was incubated for 15 to 24 hours at 37°C with orbital shaking (150 rpm) in a thermostatted air shaker. After the incubation period, an aliquot of pure methanol (no chloroform was used) was added to each tube or well to terminate the enzyme reaction. The incubated reaction mixture was freed of solvent and dried down by means of a vacuum dryer before entering the mass spectrometer. For mass spectrometry analysis, the electrospray source was operated in...

Embodiment 2

[0062]The enzymatic reaction using a substrate for Pompe disease followed by mass spectrometry analysis was carried out using the method detailed in Example 1.

[0063] Figure 8 Examples are shown in which the substrates of the invention are synthesized using α-D-glucose as the sugar moiety, for example at figure 1 Those described in , thus have specificity for the lysosomal enzyme GAA (Pompe disease). The alkyl chain was chosen to contain 10 carbons for this particular example, and therefore decanoyl-carnitine was used in the synthesis of the substrate. Similarly, the corresponding internal standard was synthesized using decanoyl-carnitine, substituting 3 hydrogens for deuterium on one of the 3 methyl groups of the carnitine moiety. Using this substrate and internal standard, dried blood spot samples from two neonates—one healthy individual and the other confirmed Pompe positive—were used as Figure 5 The method of the invention described in the treatment. Alternatively,...

Embodiment 3

[0065] Figure 9 Results of processing 12 dried blood spot samples from healthy neonates and 3 from Pompe positive neonates are shown. The reagents and methods used were Figure 6 The same as those described in . Because the internal standard is introduced at a known concentration, it is possible to quantify enzyme activity by correlating the relative abundance of product and internal standard with the concentration of the internal standard. The figure shows a scatter plot of the determined enzyme activity expressed in blood units of micromoles / hour / liter for the 15 subjects studied. From this figure, it is evident that there is a clear difference between the GAA activity of healthy neonates and that of affected neonates. The activity recorded by the three Pompe patients was negligible compared to those of healthy individuals. Note that as in Figure 6 As described in , non-enzymatic hydrolysis produces a small degree of background. The data presented here are not correc...

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 a substrate and an internal standard used in mass spectrography detection. The inventive substrate is provided which includes a substrate compound of formula A - B<1> - B<2> - B<3>: wherein A is a sugar moiety; B<1> is a linker moiety allowing the conjugation of moiety A and the remaining structure of the substrate; B<2> contains a permanently charged element such as a quaternary ammonium group so as to increase proton affinities and ionization efficiencies for mass spectrometry analysis; and B<3> of various carbon length conferring specificities to targeted enzymes. Also provided is a process to detect lysosomal diseases by contacting a sample with the inventive substrate along with an internal standard which is isotope-labeled analog of the product cleaved by a targeted enzyme upon the substrate.

Description

[0001] Cross References to Related Applications [0001] This application claims priority to US Provisional Patent Application Serial No. 60 / 781,855, filed March 13, 2006, which is incorporated herein by reference. field of invention [0002] The present invention relates to analytical reagents and mass spectrometry-based methods that use these reagents to simultaneously quantify proteins in a sample. More specifically, the invention relates to multiplex assays and reagents for quantifying lysosomal enzyme activity using mass spectrometry. Background of the invention [0003] Lysosomal storage diseases are a group of genetic conditions characterized by a deficiency of specific enzymes in the body, which results in the body's inability to break down substances. As an example, Fabry disease is a lysosomal storage disease seen in 1 in 40,000 people. It is due to a deficiency of the enzyme alpha-galactosidase, which then results in the inability of the body to break down a spe...

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): C12Q1/34G01N27/62
CPCG01N33/6848G01N33/573C12Q1/34G01N33/6893G01N2800/04
Inventor B·赛达
Owner PERKINELMER HEALTH SCIENCES INC
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