Fast method to analyse blood samples for the identification of hemoglobin variants using electron transfer dissociation

Patent metadata

Field	Value
Publication number	US-10684288-B2
Application number	US-201515128198-A
Country	US
Kind code	B2
Filing date	Mar 31, 2015
Priority date	Mar 31, 2014
Publication date	Jun 16, 2020
Grant date	Jun 16, 2020

Abstract

A method of screening or testing a sample is disclosed that comprises ionising a native human hemoglobin sample to generate parent or precursor ions, subjecting the parent or precursor ions to Electron Transfer Dissociation fragmentation so as to generate a plurality of fragment ions, mass analysing the fragment ions and determining whether or not the fragment ions include fragment ions which are indicative of a variant of hemoglobin.

First claim

The invention claimed is:

1. A method of screening or testing a sample comprising:
ionising a native human non-denatured hemoglobin sample to generate parent or precursor ions, wherein said sample is a non-denatured whole blood sample diluted with a non-denaturant, and wherein said sample has not been desalted;
subjecting said parent or precursor ions to Electron Transfer Dissociation fragmentation so as to generate a plurality of fragment ions;
mass analysing said fragment ions; and
determining whether or not said fragment ions include fragment ions which are indicative of a variant of hemoglobin.

2. A method as claimed in claim 1 , wherein the step of determining whether or not said fragment ions include fragment ions which are indicative of a variant of hemoglobin further comprises determining whether or not fragment ions having a −30.0 Da mass difference from β A C 6 fragment ions having a mass to charge ratio of 694.4 are present.

3. A method as claimed in claim 1 , wherein the step of determining whether or not said fragment ions include fragment ions which are indicative of a variant of hemoglobin further comprises determining whether or not β S C 6 fragment ions having a mass to charge ratio of 664.4 are present.

4. A method as claimed in claim 1 , wherein the step of determining whether or not said fragment ions include fragment ions which are indicative of a variant of hemoglobin further comprises determining whether or not β S C 7 fragment ions having a mass to charge ratio of 793.5 are present.

5. A method as claimed in claim 1 , wherein said variant of hemoglobin comprises the HbAS (sickle heterozygote) variant of hemoglobin.

6. A method as claimed in claim 1 , wherein said native human hemoglobin sample comprises intact non-covalently assembled tetramer of human hemoglobin.

7. A method as claimed in claim 1 , wherein said method steps are performed in vitro and are not performed on a human body.

8. A method as claimed in claim 1 , wherein said method is performed on a native human hemoglobin sample without the patient who provided said sample being present.

9. A method as claimed in claim 1 , wherein said sample is not returned to a patient.

10. A method as claimed in claim 1 , wherein said step of ionising said native human hemoglobin sample comprises ionising a sample of whole blood dissolved in a neutral buffer to generate parent or precursor ions.

11. A method as claimed in claim 10 , wherein said sample comprises ≤1000 μL, ≤500 μL, ≤100 μL, ≤50 μL or ≤10 μL of whole blood.

12. A method as claimed in claim 1 , wherein said non-denaturant is a neutral buffer comprising a phosphate buffer, a citrate buffer, an acetate buffer, a citrate-phosphate buffer or Tris-HCl.

13. A method as claimed in claim 12 , wherein said non-denaturant is a neutral buffer comprising ammonium acetate.

14. A method as claimed in claim 1 , wherein said sample is not diluted with formic acid.

15. A method as claimed in claim 1 , wherein said sample is not diluted with an organic solvent.

16. A method of screening or testing a sample comprising:
ionising a native human non-denatured hemoglobin sample to generate parent or precursor ions, wherein said sample is a non-denatured whole blood sample diluted with a non-denaturant and wherein said sample has not been desalted;
subjecting said parent or precursor ions to Electron Transfer Dissociation fragmentation so as to generate a plurality of fragment ions;
mass analysing said fragment ions and obtaining first mass spectral data;
comparing said first mass spectral data with second mass spectral data wherein said second mass spectral data relates to a hemoglobin control sample (HbAA) that has no abnormalities detected; and
determining whether or not said first mass spectral data differs from said second mass spectral data so as to indicate that said native hemoglobin sample comprises a hemoglobin variant.

17. A method as claimed in claim 16 , wherein said variant of hemoglobin comprises the HbAS (sickle heterozygote) variant of hemoglobin.

18. A method of mass spectrometry comprising a method of screening or testing a sample as claimed in claim 1 .

Assignees

• Micromass Ltd

Inventors

• Anderson Malcolm
• Brown Jeffery Mark
• Williams Jonathan

Classifications

• G01N33/721

  Haemoglobin · CPC title

• G01N33/726

  Devices · CPC title

• H01J49/0072

  by ion/ion reaction, e.g. electron transfer dissociation, proton transfer dissociation · CPC title

• G01N33/6848 Primary

  Methods of protein analysis involving mass spectrometry · CPC title

• H01J49/0031

  Step by step routines describing the use of the apparatus (H01J49/0081 takes precedence) · CPC title