Mass spectrometry-based diagnosis of hemoglobinopathies1 1 2 2 1Charlotte A. Scarff , Konstantinos Thalassinos , Nicholas Jackson , Yvonne Elliot , James H. Scrivens .

1 2Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK University Hospitals Coventry and Warwickshire NHS Trust, Coventry, UK

Hemoglobinopathies are disorders of the protein .Hemoglobin (Hb) is responsible for transporting oxygen from your lungs to the rest of the body.

hemoglobin

heme group

â-chain

á - c h a i n

The tetrameric adult human complex

hemoglobin

! 2 á-chains! 2 â-chains ! 4 heme groups

Mutations in the amino acid composition of the á-chain or the â-chain produce structural hemoglobin variants. Most cause minimal symptoms but a small proportion are life-threatening and require rapid detection so that early treatment can be given.

The clinically significant hemoglobin variants are routinely screened for, under the NHS Sickle Cell and Thalassaemia program, using electrophoresis and chromatography techniques. These techniques rely on a ‘trace-matching’ approach and therefore do not provide definitive diagnosis. If a variant is detected within a blood sample that cannot be idenified by this approach the sample is then sent for mass spectrometry or DNA analysis for conclusive characterisation.

Mass spectrometry (MS) is a method used to weigh molecules. A mass spectrometer is able to generate ions from a sample and then separate these ions based on their mass-to-charge ratio. As well as providing mass information, mass spectrometric data of peptides and proteins can be used to infer their sequence and shape.

Schematic representation of a typical mass spectrometry experiment

m/z

Inte

nsi

ty

VMASS ANALYSER

ION SOURCE

SAMPLE MASS SPECTRUM

Variant MS/MS 922.5 m/z

Control MS/MS 952.5 m/z

-30 Da

y’’2

y’’2

y’’4

y’’4

y’’3

y’’3

V H L T P E E K

V H L T P V E K

mass14800 14900 15000 15100 15200 15300 15400 15500 15600 15700 15800 15900 16000 16100 16200 16300 16400

%

0

100

1.) 10 µl of blood sample diluted in 490 µl of H O to make stock 2

solution. Stock solution diluted 10-fold in 50% acetronitrile 0.2% formic acid and analysed by mass spectrometry.

500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450m/z0

100

%

heme

a11+

a12+a13+

a14+

a15+

a16+

a17+a18+a19+

a20+

a21+

a22+

ß11+ß12+ß13+

ß14+ß15+

ß16+ß17+ß18+ß19+

ß20+

á-chain

â-chain

ág

âg

ä

+ 162 DaGlucose glycated

á-chain

ä-chain >3% indicates thalassemiaâ-

â-chain variant-30 Da

glycatedâ-chain

Indicator of diabetes

deconvoluted onto true

mass scale

2.) 100 µl of stock solution digested with the enzyme trypsin. After 30 minutes digestion diluted 10-fold in 50% acetronitrile 0.2% formic acid and analysed by mass spectrometry.

Produces a mixture of chain and tryptic peptides suitable for direct MS analysis

á- â-chain

Trypsin is a protease that cleaves protein chains after positive residues lysine (K) or arginine (R), except when they are directly followed by proline (P)

Variant tryptic digest

Control tryptic digest

-30 Da ßT11+

ßT11+

Any variant peptides present in the tryptic digest are detected by mass spectrometry.

Variant peptide detected at -30 Da from first tryptic peptide in â-chain (âT1)

Amino acid sequence V H L T P E E KâT1:

3.) The identified variant peptide is analysed by tandem mass spectrometry. The peptide of interest is selected and fragmented by collisions with a gas. The resulting fragments are analysed to determine its amino acid sequence and thus the hemoglobinopathy present.

! Definitive characterisation of hemoglobinopathy using expert- based approach for interpretation and diagnosis

! Variant identification and quantitation of glycation and delta- chain in one experiment

! Rapid identification! Capable of 24/7 operation! High initial cost but little consumables required! MS use in clinical setting already established for metabolite

screening

What are hemoglobinopathies?

Hemoglobinopathy screening

Method

Mass Spectrometry

Advantages30 mins

Variant confirmed asSickle trait b6(E®V)