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Amine-containing compound analysis methods

a compound analysis and compound technology, applied in the field of amine-containing compound analysis methods, can solve the problems of hplc having drawbacks, difficult and time-consuming derivatization step performing before analysis, and the absolute quantitation of amine-containing compounds by the above-mentioned methods can be problemati

Inactive Publication Date: 2006-08-17
DH TECH DEVMENT PTE
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  • Application Information

AI Technical Summary

Benefits of technology

[0004] The more recent use of LC / MS and MS / MS for the detection and quanititation of amine-containing compounds offers the advantage of increased sensitivity and specificity and the ability to rapidly measure multiple amine-containing compounds in one sample; however, these techniques also lack a multiplexing capability. In order to perform absolute quantitation, expensive isotopically enriched compounds are used as internal standards, which are incompatible with some tandem mass spectrometry methods. SUMMARY
[0017] Although, e.g., isotopically enriched amino acids can be used as internal standards for absolute quantitation of amino acid concentrations, one drawback of using stable isotope analogs of amino acids in MALDI mass spectrometry is that in some cases matrix related signals can interfere in the m / z region of interest of the amino acid. For example cyano-4-hydroxy cinnamic acid (CHCA) related signal at about m / z=147 can interfere with lysine and its stable isotope analog, similarity dihydroxybenzoic acid (DHB) related signal at about m / z=175 can interfere with arginine and its stable isotope analog. In various embodiments, the present teaching provide methods using isobaric tags and PDITM that facilitates reducing interference of matrix related signals with those from an internal standard or amine-containing compound of interest.

Problems solved by technology

Absolute quantitation of amine-containing compounds by the above-mentioned methods can be problematic.
For example, in order to analyze many amine-containing compounds by HPLC, a difficult and time-consuming derivatization step must be performed before analysis occurs.
In addition, HPLC has the drawbacks of long analysis times, high run-to-run deviations, a lack of multiplexing capability and non-specificity.
The more recent use of LC / MS and MS / MS for the detection and quanititation of amine-containing compounds offers the advantage of increased sensitivity and specificity and the ability to rapidly measure multiple amine-containing compounds in one sample; however, these techniques also lack a multiplexing capability.
In order to perform absolute quantitation, expensive isotopically enriched compounds are used as internal standards, which are incompatible with some tandem mass spectrometry methods.
Although, e.g., isotopically enriched amino acids can be used as internal standards for absolute quantitation of amino acid concentrations, one drawback of using stable isotope analogs of amino acids in MALDI mass spectrometry is that in some cases matrix related signals can interfere in the m / z region of interest of the amino acid.

Method used

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Examples

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example 1

Compounds from Different Sample Types

[0111] The following example illustrates the use of a variety of sample types. The teachings of this example are not exhaustive, and are not intended to limit the scope of these experiments or the present teachings

[0112] Various embodiments of the analysis of one or more amine-containing compounds from various sample types using a method of the present teachings are illustrated. In various embodiments, the one or more of amine-containing compounds of interest can originate from one or more diverse sample types, e.g, plasma, wines, proteins, peptides and amino acids in this example. In general, a sample can be processed such that compounds of interest in a sample contain one or more amine groups suitable for labeling with an isobaric tag, such as, for example, primary and secondary amines.

[0113] Referring to FIG. 3, in various embodiments, the labeling of one or more amine-containing compounds contained in a sample of plasma (block 305) compris...

example 2

Sample Preparation and Labeling with iTRAQ™ Brand Reagents

[0117] The following example illustrates examples of various embodiments preparaing and labeling one or more samples comprising one or more proteins or peptides with one or more isobaric tags using iTRAQ™ brand reagents. The teachings of this example are not exhaustive, and are not intended to limit the scope of these experiments or the present teachings.

[0118] Reduction and of Protein or Peptide Samples and Cysteine Blocking

[0119] Referring to FIG. 4A, in various embodiments, to each of at least one and up to four sample tubes, each containing between 5 and 100 μg of protein, is added 20 μL Dissolution Buffer and 1 μL Denaturant, followed by vortexing to mix (step 405). To each sample tube, 2 μL Reducing Reagent is added, followed by vortexing to mix and incubation at 60° C. for 1 hour (step 410). Spinning each sample tube is followed by addition of 1 μL Cysteine Blocking Reagent to each sample tube, then mixing by vortex...

example 3

Combining with a Standard Compound

[0130] Referring to FIG. 5, in various embodiments, the determination of the concentration of one or more amine-containing compounds in one or more samples can proceed with providing one or more amine-containing compounds, including, but not limited to peptides, polypeptides, proteins, amino acids, nitrofuran metabolites, polyamines and catecholamines. In various embodiments, an isobarically labeled standard compound (step 505) (e.g., an amine-containing compound of interest from a control sample, an amine-containing compound of interest from a sample of known concentration, etc.) is used as an internal standard (e.g., combined with) two or more isobarically labeled amine-containing test compounds to be analyzed, e.g., test compound #1 (step 510), test compound #2 (step 520), and test compound #3 (step 525).

[0131] In various embodiments, two or more of the amine containing compounds to be analyzed comprise the same amine-containing compound of int...

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Abstract

The present teachings provide methods for analyzing one or more amine-containing compounds in one or more samples using isobaric labels and parent-daughter ion transition monitoring (PDITM). In various embodiments, the methods comprise the steps of: (a) labeling one or more amine-containing compounds with different isobaric tags from a set of isobaric tags, each isobaric tag comprising a reporter ion portion; (b) combining at least a portion of each of the isobarically labeled amine-containing compounds to produce a combined sample; (c) subjecting at least a portion of the combined sample to PDITM; (d) measuring the ion signal of one or more of the transmitted reporter ions; and (e) determining the concentration of one or more of the isobarically labeled amine-containing compounds based at least on a comparison of the measured ion signal of the corresponding reporter ion to one or more measured ion signals of a standard compound.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of and priority to copending U.S. Provisional Application No. 60 / 651,734, entitled “Amine-containing Compound Analysis Methods”, filed Feb. 9, 2005, the entire disclosure of which is herein incorporated by reference.INTRODUCTION [0002] Amine-containing compounds represent important biological and medicinal chemicals. Examples of amine-containing compounds include proteins, peptides, polyamines, amino acids, catecholamines and nitrofuran metabolites. Current methods for the quantitation of amine-containing compounds include high performance liquid chromatography (HPLC) with ultra-violet (UV) fluorescent, or electrochemical detection, liquid chromatography in conjunction with mass spectrometry (LC / MS) and tandem mass spectrometry (MS / MS). [0003] Absolute quantitation of amine-containing compounds by the above-mentioned methods can be problematic. For example, in order to analyze many amine-contai...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/00
CPCG01N33/6848Y10T436/142222Y10T436/24Y10T436/173845
Inventor NIMKAR, SUBODH B.PURKAYASTHA, SUBHASISHPAPPIN, DARRYLDANIELS, SCOTT
Owner DH TECH DEVMENT PTE
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