Proteomic Detection of Beta Thalassaemia Trait Using Electrospray Mass Spectrometry-Mass Spectrometry.

Introduction Mass spectrometry has proved a valuable tool in the unequivocal characterisation of haemoglobin variants detected during routine haemoglobinopathy screening using High Performance Liquid Chromatography (HPLC) and/or isoelectric focusing. We have recently described a rapid and specific electrospray mass spectrometry-mass spectrometry (MSMS) method, using proteomic mutation targeting, for simultaneous detection of the clinically significant haemoglobinopathies; haemoglobin (Hb)S, HbC, HbE, HbD^Punjab and HbO^Arab. Here we report additional transitions within the 1min cycle time, using delta chains as a surrogate HbA₂, with the objective of detecting beta thalassaemia trait.

Method Blood samples are digested using trypsin, which results in specific cleavage of globin chains at peptide bonds adjacent to lysine and arginine residues, to produce a series of well-characterised peptides. There is strong amino acid sequence homology between the beta and delta chains of haemoglobin, however tryptic digestion produces series of 15 (T1–T15) and 16 (T1–T16) peptides, for beta and delta chains respectively, with differences in T2, T3, T5, T10, T12, T13 and T14. In this report we have investigated the diagnostic utility of T2: beta sequence SAVTALWGK, average mass 932.1 daltons, delta sequence TAVNALWGK, average mass 959.1 daltons. In multiple reaction monitoring mode the respective T2 [M+2H]²⁺ ions, m/z 466.8 and 480.3, are selected, fragmented, and the singly charged ions of the informative peptide fragments, VTALWGK and VNALWGK, detected.

Results The percentage area ratio of delta/(beta+delta) was calculated and compared with the classical percentage HbA₂ value obtained by HPLC. 66 samples with HbA₂ values within our normal range (mean 2.7%, range 1.8–3.4), and 58 with HbA₂ values indicative of beta thalassaemia trait (5.2%, 4.2–7.9) were analysed by MSMS and the corresponding delta/(beta+delta) ratios were 1.7%, 0.9–2.3 and 3.4%, 2.5–6.0. In addition, in 2 further HbS beta thalassaemia zero compound heterozygote samples confirmed by beta gene sequencing the ratios were 4.1% and 3.7%.

Conclusion This relatively small study provides evidence that beta thalassaemia trait may be diagnosed by MSMS and represents another step towards comprehensive MSMS population screening for clinically significant haemoglobinopathies.