Case Study One: Patient A

The HPLC trace from the analysis of a blood sample from patient A is shown below in Figure 5.4.

Figure 5.4: HPLC trace from TOSOH HLC-723 HbG7 analyser for blood sample from patient A. Retention time window for Hb S is shown in blue.

The HPLC trace shows the presence of an additional peak with a retention time (rt) of approximately 5.8 minutes. The variant responsible for this peak represents approximately 15.8 % of the total hemoglobin. This peak elutes just inside the Hb S retention time window but is not Hb S. The presence of a variant at such a low

percentage indicates that it is probably an α-chain variant. Αs there are four α-globin

genes, a mutation within one of these genes usually leads to the expression of an α- chain variant 10-25 % of the time. A β-chain variant is usually expressed at 40-50 %. The identification of this variant cannot be determined by HPLC or IEF analysis.

ESI-MS analysis of this blood sample followed by spectral deconvolution was used to investigate whether the variant was in the α-or β-chain. The α-chain was first used as an internal data calibrant. The deconvoluted spectrum obtained after linear

correction with the α-chain is shown below in Figure 5.5. This shows an error in β- chain mass measurement of +0.28 Da. This alone would suggest the presence of a +1

Figure 5.5: Deconvoluted spectrum obtained after ESI-MS analysis of blood sample from

patient A with the α-chain used as an internal calibrant.

As the calibration of the data was performed with the α-chain, however, the α-chain mass measurement was corrected to normal. It was therefore possible that the variant was a -1 Da α-chain variant instead. The intact MS analysis, alone, cannot confirm whether the variant is a -1 Da α-chain variant or a +1 Da β-chain variant. Electrophoretic charge change information or MS analysis of the tryptic digest is required to confirm this.

The HPLC trace showed that the variant had a positive polarity change and so it was

inferred that the variant was an α-chain variant. A single amino acid mutation which produces a zero or negative mass change gives a positive or zero electrophoretic charge change and the converse is also true (Rai et al. 2003). The positive β-chain mass change originally observed, together with the positive polarity change observed in the HPLC trace, indicated, therefore, that the variant was actually in the α-chain.

The β-chain was used to re-calibrate the data file prior to deconvolution. The

resulting deconvoluted spectrum is shown in Figure 5.6. The α-chain mass

measurement (after linear correction with the β-chain) was -0.26 Da from the normal

α-chain mass which indicated that a -1 Da α-chain was present. If HPLC information was not available, or this analysis was being conducted in an automated fashion,

chain +0.28 Da - β 16171.18 15867.52 15031.76 15126.38 100 0 % 16750 16500 16250 16000 15750 15500 15250 15000 mass

intact MS analysis would indicate the presence of a -1 Da α-chain variant or a +1 Da

β-chain variant. In this case the sample would be subjected to further analysis as the presence of a variant was indicated and this further analysis would be used to determine the identification of the variant.

Figure 5.6: Deconvoluted spectrum obtained after ESI-MS analysis of blood sample from

patient A with the β-chain used as an internal calibrant.

The blood sample from patient A was subsequently subjected to tryptic digestion and the digested sample analysed by means of ESI-MS. The spectrum obtained showed the presence of an additional peak at 999.17 m/z(Figure 5.7). The additional peak at 999.17 m/zwas from a triply-charged ion. This corresponded to that which would be produced from a peptide 1 Da smaller than the 9th tryptic peptide of the α-chain

(αT9). This suggested that the α-chain variant present was the result of a single amino acid mutation within αT9(62-90), which produced a -1 Da mass change. There are four possible amino acid changes which result in a -1 Da mass shift (D→N, E→Q, N→I/L , E→K) and six positions within αT9 where one of these changes could have occurred (Figure 5.8). This information combined with the positive electrophoretic charge change observed in the HPLC trace would suggest that the mutation was D→N but does not give the exact mutation site.

0.26 Da - chain - α 16171.16 15867.24 14915.56 15126.12 100 0 % 16750 16500 16250 16000 15750 15500 15250 15000 mass

Figure 5.7:ESI-spectra of tryptic digest of blood sample from patient A (red) and control (green). Inset spectra show the 999-1002 m/zregion in greater detail. A peak is observed at 999.17 m/zin the sample which is not observed in the control.

VADALTNAVAHVDDMPNALSALSDLHAHK

N

I/L

NN

I/L

N

αT9(62-90)

Hb G-Norfolk Hb G-Waimanalo

Hb G-Pest Hb Matsue-Oki

Figure 5.8: Schematic of αT9, amino acids 60-92 in the α-chain (shown in single-letter code). The potential single amino acid mutations which would result in a -1 Da mass change are illustrated.

The mutation site was confirmed by MS/MS analysis. Spectra obtained for MS/MS analysis on 999.5 (±0.5) m/z ([αT9 + 3H]3H+) from a control and the sample are shown in Figure 5.9. Presence of fragment ions at -1 Da for y”6 and larger y” ions

but not for y”5 confirms that the mutated site is α85(Asp→Asn). A search of HbVar

showed that the variant present was Hb G-Norfolk. This variant is found in a few families in England and Canada and is associated with increased oxygen affinity.

a.)

b.)

Figure 5.9:a.) MS/MS spectra of 999.5 m/z from variant sample (red) and control (green). Additional fragment ions are observed in the sample spectrum at -1 Da from the y”6 ion and larger y” ions. These additional fragment ions confirm the identity of the variant present as α85(Asp→Asn). b.) Expected fragment ions for 999.5 m/z are shown.

m/z Sample MS/MS 999.5 1 Da - 1 Da - m/z Control MS/MS 999.5 y”6 y”6 y”7 y”7 y”5 y”5