Mass spectrometry-based diagnosis of hemoglobinopathies
1
1
2
2
1
Charlotte A. Scarff , Konstantinos Thalassinos , Nicholas Jackson , Yvonne Elliot , James H. Scrivens .
1
Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK 2University Hospitals Coventry and Warwickshire NHS Trust, Coventry, UK
What are hemoglobinopathies?
Method
Hemoglobinopathies are disorders of the protein hemoglobin.
Hemoglobin (Hb) is responsible for transporting oxygen from your
lungs to the rest of the body.
1.) 10 µl of blood sample diluted in 490 µl of H2O to make stock
solution. Stock solution diluted 10-fold in 50% acetronitrile 0.2%
formic acid and analysed by mass spectrometry.
á-chain
heme group
Variant tryptic digest
The tetrameric adult
human hemoglobin
complex
a 19+a 18+
20+
a
a 17+
a 15+
%
a 22+
ß19+ß18+
ß17+ß16+
!
550
600
650
700
750
800
850
900
950
1000
Amino acid sequence âT1: V H L T P E E K
ßT11+
a 14+
ß15+
ß14+
a 13+
a 12+
ß13+ ß12+
a 11+ ß11+
ß20+
0
500
Control tryptic digest
deconvoluted
onto true
mass scale
a 16+
a 21+
â-chain
ßT11+
-30 Da
heme
100
2 á-chains
! 2 â-chains
! 4 heme groups
Variant peptide detected at -30 Da from
first tryptic peptide in â-chain (âT1)
1050
1100
1150
1200
1250
1300
1350
1400
1450
m/z
100
â-chain
Mutations in the amino acid composition of the á-chain or the âchain 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.
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.
â-chain
variant
-30 Da
á-chain
Hemoglobinopathy screening
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
Schematic representation of a typical
mass spectrometry experiment
15000
15100
15200
15300
Variant confirmed as
Sickle trait b6(E®V)
ä-chain >3%
indicates
â-thalassemia
15500
15600
15700
15800
y’’2
y’’3
ä
15400
-30 Da
glycated
â-chain
glycated
á-chain
ág
14900
15900
âg
Control MS/MS 952.5 m/z
V
V H LT PE E K
y’’4
16000
16100
16200
16300
mass
16400
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.
y’’2
y’’3
30 mins
Advantages
!
m/z
MASS
ANALYSER
V H LT PV E K
y’’4
Indicator of
diabetes
Intensity
MASS
SPECTRUM
ION
SOURCE
+ 162 Da
Glucose
0
14800
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.
SAMPLE
%
Variant MS/MS 922.5 m/z
Produces a mixture of á-chain and â-chain
tryptic peptides suitable for direct MS analysis
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)
Any variant peptides present in the tryptic digest are detected by
mass spectrometry.
!
!
!
!
!
Definitive characterisation of hemoglobinopathy using expertbased approach for interpretation and diagnosis
Variant identification and quantitation of glycation and deltachain 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