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

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.

Claims

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 .sup.AC.sub.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 .sup.SC.sub.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 .sup.SC.sub.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 L, 500 L, 100 L, 50 L or 10 L 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various embodiments will now be described together with other arrangements given for illustrative purposes only, by way of example only, and with reference to the accompanying drawings in which:

(2) FIG. 1 shows a mass spectrum of native human hemoglobin;

(3) FIG. 2 shows a fragmentation spectrum of the fragment ions obtained from subjecting [M+17H].sup.17+ parent ions of human hemoglobin to Collision Induced Dissociation;

(4) FIG. 3A shows an Electron Transfer Dissociation mass spectrum of [M+18H].sup.18+ parent ions of human hemoglobin showing beta-chain annotation and FIG. 3B shows the same Electron Transfer Dissociation mass spectrum of [M+18H].sup.18+ parent ions from human hemoglobin but showing alpha-chain annotation;

(5) FIG. 4 shows partial mass spectra of normal (HbAA) and the sickle variant (HbAS) of hemoglobin obtained according to an embodiment and wherein the peak at mass to charge ratio 664.4 having a mass difference of 30 Da relative to the fragment mass peak at 694.4 is indicative that the hemoglobin sample comprises the sickle variant of hemoglobin; and

(6) FIG. 5 shows in greater detail partial mass spectra of normal (HbAA) and the sickle variant (HbAS) of hemoglobin.

DETAILED DESCRIPTION

(7) A conventional approach to analysing hemoglobin will first be described.

(8) Normal adult human hemoglobin (Hb) exists as a non-covalently assembled tetramer consisting of two alpha chains (MW 15,126.4) and two beta-chains (15,867.2) in which each chain is associated with a heme group (MW 616.5). The average molecular weight of the intact assembly is 66,453.2. The primary function of hemoglobin is to deliver oxygen to the organs of the body. Structural abnormalities within the sequence of one of these chains can affect the overall function of the assembled hemoglobin tetramer.

(9) In adult human hemoglobin approximately 1000 alpha- and beta-chain abnormalities (variants) have been described and many more are possible.

(10) Hemoglobin variants are generally caused by a single base mutation in a globin gene. Some variants are clinically significant whilst many function normally. Knowledge of how each type of change specifically alters the function is important in understanding how hemoglobin works as well as for treating diseases caused by hemoglobin variants.

(11) FIG. 1 shows a parent ion mass spectrum of native human hemoglobin showing multiply charged ions detected ranging from [M+15H].sup.15+ to [M+20H].sup.20+.

(12) FIG. 2 shows a fragmentation mass spectrum obtained by subjecting multiply charged precursor ions [M+17H].sup.17+ of human hemoglobin to Collision Induced Dissociation fragmentation. Following ejection of an alpha- or beta-chain sub-unit, mixed trimeric species (.sub.2 and .sub.2) are detected with and without the heme group. Sub-unit loss is observed following Collision Induced Dissociation and little, if any, sequence-specific information is obtained unless more complex experiments such as MS.sup.3 or higher are performed.

(13) An embodiment will now be described.

(14) The embodiment described below relates to methodologies involving the Electron Transfer Dissociation analysis of native hemoglobin clinical samples in order to provide a clinical diagnosis.

(15) Electrospray ionisation combined with Electron Transfer Dissociation of selected multiply charged precursor ions produced from the intact non-covalently assembled tetramer (.sub.2.sub.2+4haem) of human hemoglobin is able to rapidly identify examples of human hemoglobin variants.

(16) Electron Transfer Dissociation fragmentation method provides a rapid non-ergodic reaction of selected positive multiply charged ions with radical anions and causes extensive cleavage of the peptide backbone.

(17) FIGS. 3A and 3B show an Electron Transfer Dissociation mass spectrum of multiply charged precursor ions [M+18H].sup.18+ from human haemoglobin obtained using 4-nitrotoluene as the ETD reagent ions. The mass spectra shown in FIGS. 3A and 3B are the same but the mass spectra have been separately annotated with alpha- and beta-chain notation.

(18) It is clear that extensive sequence-specific information is provided through the use of Electron Transfer Dissociation fragmentation compared to Collision Induced Dissociation fragmentation and highlights the ability of Electron Transfer Dissociation and subsequent mass analysis of human hemoglobin in order to identify or detect hemoglobin variants.

(19) FIG. 4 shows an example of the identification of HbAS (sickle heterozygote) using native or natural hemoglobin which is present in a blood sample and top-down Electron Transfer Dissociation mass spectrometry obtained using 4-nitrotoluene as the ETD reagent ions. The mass spectral data may be compared with a normal hemoglobin control (HbAA) that has no abnormalities detected.

(20) The hemoglobin sickle variant (HbAS or HbSS), 6Glu (E).fwdarw.Val (V), can be identified by various phenotypic methods. Identification can be questioned when the sickle variant in a heterozygote is significantly different from its normal value (40%). Therefore, DNA analysis or confirmation by mass spectrometry is required.

(21) Mass spectrometry is fast and using the method according to an embodiment enables protein sequence information to be rapidly used to determine HbAS or HbSS for example. Other clinically significant and or innocuous variants can also be identified using the technique according to an embodiment.

(22) FIG. 4 shows the partial mass spectra obtained from native top-down Electron Transfer Dissociation mass spectrometry of a normal hemoglobin (HbAA) and a sickle variant hemoglobin heterozygote (HbAS).

(23) FIG. 5 shows in greater detail the partial mass spectra highlighting a 30.0 Da mass difference at position c6 in the beta-chain from normal. This data precisely shows the ability of hemoglobin phenotyping using the method according to an embodiment.

(24) The samples were analysed as received and diluted for Electrospray ionisation-mass spectrometry analysis to a concentration of 10 M (100 mM ammonium acetate) without any prior work up or de-salting procedures.

(25) Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the present invention as set forth in the accompanying claims.