Chapter 4: A Quantitative and Conformational Study of the
5.3.2 Case Study Two: Patient B
The HPLC trace from the analysis of a blood sample from patient B is shown below in Figure 5.10.
Figure 5.10: HPLC trace from TOSOH HLC-723 HbG7 analyser for blood sample from patient B. Retention time windows for Hb D, Hb S and Hb C are shown in green, blue and red respectively.
The HPLC trace showed the presence of three additional peaks with retention times in the Hb D, Hb S and Hb C retention windows respectively. The presence of four main peaks in the HPLC trace suggested that two variants were present within this sample.
The deconvoluted spectrum obtained following ESI-MS analysis of the blood sample from patient B is shown below in Figure 5.11. Four main peaks are seen in the spectrum at masses 15126.38, 15140.13, 15837.34 and 15867.12 Da. The peaks are
identified as α-chain, α-chain variant (+14 Da, α+14), β-chain variant (-30 Da, β-30)
and β-chain respectively. The mass measurement of the β-chain in this case is not as accurate as would be expected. The mass error of -0.12 Da would ordinarily be
expected to represent the presence of a β-chain variant at a low percentage. There are
of a -30 Da β-chain variant. The other β-chain present must be normal. If an additional variant was present the HPLC trace for this sample would be different. There would be further additional peaks, representing variant dimers, and no peak
corresponding to the elution of normal αβ-dimer.
mass 14800 15000 15200 15400 15600 15800 16000 16200 16400 16600 % 0 100 15126.38 15038.04 15867.12 15140.13 15837.34 15836.36 15497.68 15802.06 15969.78 16173.10 βchain βchain - 30Da α-chain α-chain + 14 Da
Figure 5.11: Deconvoluted spectrum obtained after ESI-MS analysis of blood sample from patient B. The α-chain was used as an internal calibrant. The main peaks within the spectrum
are identified as α-chain, α-chain variant (+14 Da), β-chain variant (-30 Da) and β-chain.
When additional peaks are observable in the deconvoluted mass spectrum the mass measurement of individual peaks is often not within ±0.1 Da. The error in mass measurement is increased to ± 0.25 Da here. This does not cause a problem as the observation of an error in mass measurement greater than 0.1 Da is always investigated further. The percentage of α-chain variant present within the sample was
estimated, by integration of the areas under the peaks corresponding to α-chain and
α-chain variant within the deconvoluted spectrum, to be 51 %. The β-chain variant was estimated to represent 30 % of the total β-chain. The percentage of α-chain
variant present was higher than would normally be expected if only one α-gene locus
was mutated. This suggested the possibility that this patient had α-thalassemia. The blood sample from patient B was subsequently subjected to tryptic digestion and the digested sample analysed by ESI-MS.
The spectra obtained showed the presence of some additional peaks in comparison to a control (Figures 5.12 and 5.15). Additional singly-charged ions were observed at 717.4 and 922.5 m/zand an additional triply-charged ion was observed at 771.4 m/z. The ion at 922.5 m/z corresponded to that which would be produced by a peptide 30 Da smaller than the 1st tryptic peptide of the β-chain (βT1). There are five single amino acid substitutions which can result in a -30 Da mass change (T→A, W→R, S→G, M→T and E→V) and three positions in βT1 where one of these substitutions could have occurred (Figure 5.13).
βT1
1+βT1
1+ (βT1 – 30 Da)1+Sample
Control
Figure 5.12: ESI-MS spectra of tryptic digest of blood sample from patient B (red) and a control (green). An additional ion is observed within the spectrum for the patient’s sample at 922.5 m/z. This corresponds to (βT1 -30 Da)1+.
MS/MS analysis of 922.5 m/z in comparison to 952.5 m/z (βT11+) was used to
confirm the identification of the β-chain variant present (Figure 5.14). MS/MS
analysis showed that the mutation was β6(E→V) (βS
V H L T
P E E
K
βT1(1-8)
A
V V
Hb S
Figure 5.13:Schematic of βT1, amino acids 1-8 in the β-chain (shown in single-letter code). The potential single amino acid mutations which would result in a -30 Da mass change are illustrated.
Figure 5.14: MS/MS spectra for 922.5 m/z from variant sample (red) and 952.5 m/z from control (green) with expected fragment ions for 952.5 m/zshown.
H+ [M + H] H+ [M + H] Da 30 - y”2 y”2 y”4 y”4 m/z 952.5 Control MS/MS m/z 922.5 Sample MS/MS
The α-chain variant with a +14 Da mass change could have been produced by one of five possible single amino acid substitutions (N→K, D→E, G→A, S→T or V→I/L). As two other additional peptides need to be accounted for within the ESI-MS spectra for the tryptic digest (Figure 5.15 below) a N→K mutation is most likely. This mutation produces an additional trypsin cleavage site and so would produce two new
peptides. The α-chain sequence was then analysed to determine the new peptides which would be produced by a single N→K mutation. It was found that the
additional peptides produced could be explained by an α68(N→K) (Figure 5.16). The α-chain variant was thus identified as corresponding to Hb G-Philadelphia (αG).
Sample
Control
Additional peptides 717.41+ 771.43+Figure 5.15: ESI-MS spectra of tryptic digest of blood sample from patient B (red) and a control (green). An additional singly-charged ion is observed at 717.4 m/zand an additional triply-charged ion at 771.4 m/zwithin the spectrum for the patient’s sample.
VADALTN AVAHVDDMPNALSALSDLHAHK
αT9(62-90)
K
VADALTK
AVAHVDDMPNALSALSDLHAHK
717.41+ 771.43+
Figure 5.16: Schematic of αT9, amino acids 60-92 in the α-chain. The tryptic peptides
produced for the mutant α68(N→K) are shown.
This patient has Hb S and Hb G-Philadelphia. Hb G-Philadelphia in Black populations is present with the 3.7 kilobase deletion alpha-thal-2 (-αG/αα); the quantity of Hb G in such individuals varies between 30 and 35 %. When this deletion occurs in trans (-αG/-α) the quantity of Hb G is increased to approximately 45 %. These patients have a distinct microcytosis and hypochromia. The high percentage of Hb G-Philadelphia, estimated by the MS analysis, suggested that this
individual may have this form of α-thalassemia. With the variants present identified, the four main peaks present in the HPLC trace could also be assigned. They
represented the elution of, from left to right, αβ dimer, αGβ dimer, α βSdimer and αG βS
dimer respectively. The relative intensities that these peaks were observed at within the HPLC trace could now be used to calculate the relative intensities of the variant chains within the sample. Calculations based upon the HPLC analysis showed that approximately 31 %of the β-chain variant was present and 47 % of the
α-chain variant was present. The percentages estimated by the mass spectrometric analysis of 30 % and 51 % respectively are in good agreement with these measurements. The high percentage of Hb G-Philadelphia observed strongly suggests that this patient has trans alpha-thal-2. In this case, mass spectrometric analysis has been used successfully to provide identification of a clinically important variant.