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Methods and mixtures pertaining to analyte determination using electrophilic labeling reagents

an electrophilic labeling and analyte technology, applied in the field of methods and mixtures pertaining to analyte determination using electrophilic labeling reagents, can solve the problems of difficult or impossible detection of reporter fragments above background noise, difficult or time-consuming, and difficulty in interpreting cid spectra to assemble the amino acid sequence of the parent peptide de novo,

Inactive Publication Date: 2004-11-04
DH TECH DEVMENT PTE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Likewise, because of over-enrichment (undesired enrichment) and because of natural isotopic abundance, there can be impurities of greater mass.
By "does not substantially sub-fragment" we mean that fragments of the reporter are difficult or impossible to detect above background noise when applied to the successful analysis of the analyte of interest.
Interpreting such CID spectra to assemble the amino acid sequence of the parent peptide de novo is challenging and time-consuming.

Method used

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  • Methods and mixtures pertaining to analyte determination using electrophilic labeling reagents
  • Methods and mixtures pertaining to analyte determination using electrophilic labeling reagents
  • Methods and mixtures pertaining to analyte determination using electrophilic labeling reagents

Examples

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example 1

Synthesis Of Morpholine Acetic Acid

[0205] Bromoacetic acid (2 g, 14.4 mole) was dissolved in tetrahydrofuran (50 mL) and added dropwise to a stirred solution of morpholine (3.76 g, 43.2 mole) in tetrahydrofuran (THF, 20 mL). The solution was stirred at room temperature for three days. The white solid (4.17 g) was filtered, washed with THF (100 mL), and recrystallised from hot ethanol (EtOH), Yield: 2.59 g: IR:1740 cm-1. For the two different isobaric versions of morpholine acetic acid, either bromoacetic-1-.sup.13C acid (Aldrich PN 27,933-1) or bromoacetic-2-.sup.13C acid (Aldrich PN 27,935-8) was substituted for bromoacetic acid.

example 2

Synthesis Of Morpholine Acetic Acid N-Hydroxysucciniimide Ester

[0206] Dimethylformamide (dry, 1.75 g, 0.024M) was dissolved in tetrahydrofuran (dry, 30 mLs). This solution was added dropwise to a stirred solution of thionyl chloride (2.85 g, 0.024M) dissolved in tetrahydrofuran (dry, 20 mLs) and cooled in an ice bath. After complete addition and 30 minutes on ice, the ice bath was removed and solid N-hydroxysuccinimide (2 g, 0.017 M) was added (which completely dissolved) immediately followed by solid pre-powdered morpholine acetic acid [or -1-.sup.13C or -2-.sup.13C morpholine acetic acid] (3.64 g, 0.016M). The morpholine acetic acid dissolved slowly giving a homogeneous solution that rapidly became cloudy. The reaction was left vigorously stirring over night at room temperature. The white solid was washed with tetrahydrofuran and dried under vacuum, weight 3.65 g (67%), IR spectrum 1828.0 cm-1,1790.0 cm-1,1736.0 cm-1.

example 3

Analyte Determination and Relative Quantitation in Two Samples

[0207] 100 pmole amounts of freeze-dried Glu-Fibrinopeptide B (Sigma) were reacted with 200 .mu.l of freshly-made 2% w / v solutions of either I or II (See: FIG. 1A for structure and Examples 1 & 2 for preparation) in ice-cold 0.5M MOPS buffer (pH 7.8 with NaOH) for 30 minutes on ice. The reaction was terminated by the addition of TFA to 0.5% v / v final concentration. The modified peptides were then mixed in various pre-determined proportions to approximately cover the range 1:10 to 10:1 of the differentially labeled peptides. Each peptide mixture was individually purified by reverse-phase de-salfing using a Millipore C18 Zip-Tip. Excess reagent and buffer do not retain on the reverse-phase packings, and were thus efficiently removed prior to MS analysis. The mixtures (0.5 .mu.l) were then spotted onto a MALDI target plate, over-spotted with 0.5 .mu.l of 1% w / v .alpha.-cyano cinnamic acid in 50% aqueous acetonitrile and each...

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Abstract

This invention pertains to methods, mixtures, kits and / or compositions for the determination of analytes by mass analysis using unique labeling reagents or sets of unique labeling reagents. The labeling reagents can be isomeric or isobaric and can be used to produce mixtures suitable for multiplex analysis of the labeled analytes.

Description

[0001] This application is related to U.S. Ser. No. 10 / ______ entitled: "Methods And Mixtures Pertaining To Analyte Determination" and filed on this Jan. 27, 2004, incorporated herein by reference. This application is related to U.S. Ser. No. 10 / ______ entitled: "Compositions And Kits Pertaining To Analyte Determination" and filed on this Jan. 27, 2004, incorporated herein by reference. This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60 / 443,612, filed on Jan. 30, 2003, incorporated herein by reference.[0002] This invention pertains to the field of analyte determination by mass analysis.[0003] FIG. 1A illustrates the reaction of an analyte with two different isobaric labeling reagents (e.g. compounds I and II).[0004] FIG. 1B illustrates the fragmentation of the labeled analyte illustrated in FIG. 1A to thereby produce reporter moieties (e.g. compounds VII and VIII as signature ions) of different masses from the isobarically labeled analytes.[0005] ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A23L11/20B01L99/00A61K51/04C07D207/46C07D207/48C07D295/06C07D401/12C07D413/12C12Q1/68G01NG01N1/00G01N24/00G01N27/447G01N27/62G01N30/00G01N31/00G01N33/58G01N33/68
CPCC07D401/12G01N33/532G01N33/6842G01N33/6848G01N2458/15Y10T436/24C07D207/46Y10T436/142222Y10T436/147777Y10T436/145555A61K51/04
Inventor PAPPIN, DARRYL J.C.BARTLET-JONES, MICHAEL
Owner DH TECH DEVMENT PTE
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