Quantitation of structural isomers using MALDI MS/MS

Patent metadata

Field	Value
Publication number	US-9659759-B2
Application number	US-201415024104-A
Country	US
Kind code	B2
Filing date	Sep 25, 2014
Priority date	Sep 25, 2013
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

The present invention relates to a quantitative determination of structural isomers implicated in diseases, the said process is completely independent of chromatographic separation, internal standard, and labeled isotopic references. Further, the present invention provides a diagnostic kit for quantitative determination of structural isomers.

First claim

We claim:

1. A method for quantitatively determining structural isomers comprising:
a) subjecting an equimolar mixture containing the reference isomers to ionization and selecting precursor ions having a mass to charge ratio in an m/z range between 1 to 40000 (z=1) followed by fragmentation to generate product ions that are both common and unique in nature to the reference isomers;
b) selecting product ions that are common to the isomer pairs to normalize the unique product ions followed by measuring the peak area/peak intensity ratios of the unique and common product ions to generate equimolar concentration response curves for each individual isomer pair as a function of equimolar ratios; and
c) subjecting a sample containing unknown quantities of the isomers/isomer pairs in context to ionization and selecting precursor ions having a mass to charge ratio in an m/z range between 1 to 40000 (z=1) followed by fragmentation to generate product ions that are both common and unique in nature as in step (a); measuring the unique to common product ion ratio(s), and determining the corresponding equimolar ratio(s) of the isomer pairs present based on the equimolar concentration response curve(s) generated in step (b).

2. The method according to claim 1 , wherein the common product ion production is achieved in the ionization step of (c) of claim 1 where needed by adding a known predetermined quantity of a common product ion producing non endogeneous synthetic isomer(s) to a sample containing unknown quantities of one or more of the isomers/isomer pairs in context followed by ionization and selecting precursor ions having a mass to charge ratio in an m/z range between 1 to 40000 (z=1) followed by fragmentation to generate product ions that are both common and unique in nature as in step (a); measuring the unique to common product ion ratio(s), and determining the corresponding equimolar ratio(s) of the isomer pairs present based on the equimolar concentration response curve(s) generated in step (b).

3. The method according to claim 1 , wherein the equimolar reference mixture is selected from a single pair of isomers and/or multiple pairs of different isomers mixed together to make a single or several sets of reference equimolar mixture(s).

4. The method according to claim 1 , wherein the analyte is subjected to MALDI MS/MS or LDI MS/MS analysis.

5. The method according to claim 1 , wherein the common product ions are from the analyte or a different analyte fragmenting together with analyte.

6. The method according to claim 1 , wherein the peak area/peak intensity ratios of unique and common product ions to generate concentration response curves are based on the ASCII files.

7. The method according to claim 1 , wherein the isomers are selected from the group consisting of asymmetric dimethyl arginine (ADMA) and symmetric dimethyl arginine (SDMA); leucine and isoleucine; methyl malonic acid (MMA) and succinic acid (SA); bilirubin and lumirubin and the like.

8. The method according to claim 1 , wherein the sample is a biological sample selected from urine, blood sample, blood plasma and RBC from patient samples or any other such sources of the isomers.

9. The method for quantitatively determining structural isomers as claimed in claim 1 , for use in diagnostic kit for determination of structural isomers comprising:
a) equimolar mixture of reference standards of isomer(s) pairs in vials and/or as dried precoated spots on a sample analysis plate,
b) a common product ion producing non endogeneous synthetic isomer (s)
c) a CD with algorithm for determining the isomers quantitatively, and
d) a detailed protocol to achieve this using the former together.

10. The method for quantitatively determing structural isomers as claimed in claim 9 , the diagnostic kit comprising:
a) a series of mixture of isomers in varying ratios;
b) three quality control samples in plasma or any such fluid from biological source or synthetically simulated and standards;
c) solutions/buffers (labeled as Buf1-Buf3); and/or
d) common product ion producing non endogeneous synthetic isomer(s)
e) a disposable MALDI target plate containing dried spots of Clinical Isomer Pairs (CIP-ADMA/SDMA, Leu/Ile and MMA/SA) mixture along with space for samples to be analyzed; and
f) software on a CD and instructions to use the kit contained in a Product Information Sheet.

11. A method of diagnosing a disease caused by altered concentrations of isomer ratio by measuring the ratio of the isomers in a biological sample, which process comprises:
a) isolating and providing a biological sample from a subject;
b) subjecting an equimolar mixture containing the reference isomers to ionization and selecting precursor ions having a mass to charge ratio in an m/z range between 1 to 400000 (z=1) followed by fragmentation to generate product ions that are both common and unique in nature to the reference isomers;
c) selecting product ions that are common to the isomer pairs to normalize the unique product ions followed by measuring the peak area/peak intensity ratios of the unique and common product ions to generate equimolar concentration response curves for each individual isomer pair as a function of equimolar ratios; and
d) subjecting a sample containing unknown quantities of the isomers/isomer pairs in context to ionization and selecting precursor ions having a mass to charge ratio in an m/z range between 1 to 40000 (z=1) followed by fragmentation to generate product ions that are both common and unique in nature as in step (a); measuring the unique to common product ion ratio(s), and determining the corresponding equimolar ratio(s) of the isomer pairs present based on the equimolar concentration response curve(s) generated in step (b)
wherein altered ratio of isomers in the biological sample compared to the reference is indicative that the subject has, or is at a risk of developing a disease characterized by altered ratio of the isomer.

12. The method according to claim 11 , wherein the common product ion production is achieved in the ionization step of (c) of claim 11 where needed by adding a known predetermined quantity of a common product ion producing non endogeneous synthetic isomer(s) to a biological sample isolated from a human subject or other non human biological specimen containing unknown quantities of one or more of the isomers/isomer pairs in context followed by ionization and selecting precursor ions having a mass to charge ratio in an m/z range between 1 to 40000 (z=1) followed by fragmentation to generate product ions that are both common and unique in nature as in step (a); measuring the unique to common product ion ratio(s), and determining the corresponding equimolar ratio(s) of the isomer pairs present based on the equimolar concentration response curve(s) generated in step (b).

13. The method of diagnosing a disease according to claim 11 , wherein the disease is a cardiovascular disease or a chronic kidney disease, liver disorders, maple syrup urine disease, diabetes, obesity, methylmalonic acidemia, vitamin B12 deficiency and pernicious anemia.

Assignees

• Council Scient Ind Res
• Council Scient Ind Res

Inventors

• Panchagnula Venkateswarlu
• Bhattacharya Nivedita
• Ghanate Avinash Dattatraya

Classifications

• H01J49/164 Primary

  Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI] (sample holders H01J49/0418) · CPC title

• G06F19/70

  Physics · mapped topic

• H01J49/004

  Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title