Hemoglobin (Hb) protein is made up of four polypeptide chains, with over 1000 Hb structural variants currently classified.1 Though some variants are benign, other variants, such as those responsible for thalassemias and sickle cell, can cause serious complications if they are not diagnosed early in life. In the UK and U.S., newborn-screening programs are routinely used to screen for serious Hb variants.
Edwards et al.2 demonstrated the effectiveness of using a top-down proteomics approach to screen for common Hb variants, HbS, HbC, and HbD, present in dried blood spots. This method did not require lengthy digestion steps used in the traditional bottom-up approach. With the positive results of those experiments, this group has now published the results of experiments identifying unknown Hb variants using high-resolution mass spectroscopy.3
To identify unknown Hb variants, dried blood spot samples from newborn patients were obtained and processed normally using cation exchange high-performance liquid chromatography (ceHPLC) and isoelectric focusing (IEF). After processing, six samples were determined to be Hb variants with unknown identities.
To diagnose variants, collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. A Triversa Nanomate chip-based electrospray device (Advion), coupled to an Orbitrap Velos ETD mass spectrometer (Thermo Scientific), were used to analyze the Hb variants. MS spectra were analyzed using Xcalibur 2.10 software (Thermo Scientific).
The results of these experiments classified four of the six samples as β-chain variants: Two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the α-chain variant heterozygous Hb Phnom Penh. Interestingly, analysis of the sixth sample revealed that it did contain a variant, which was either caused by mislabeling by the original possessing protocol or may have contained an isomeric substitution.
While this application of top-down proteomics did successfully identify unknown variants in Hb, the protocol and manual analysis was time consuming and would be highly inefficient in a clinical setting. The development of more data analysis software and MS/MS databases could enhance the value of this approach in a clinical setting.
1. Giardine, B., et al. (2007) ‘HbVar database of human hemoglobin variants and thalassemia mutations: 2007 update‘, Human Mutation, 28 (2), (pp. 206)
2. Edwards, R.L., et al. (2011) ‘Hemoglobin variant analysis via direct surface sampling of dried blood spots coupled with high-resolution mass spectrometry‘, Analytical Chemistry, 83 (6), (pp. 2265–2270)
3. Edwards, R.L., et al. (2012) ‘Top-down proteomics and direct surface sampling of neonatal dried blood spots: diagnosis of unknown hemoglobin variants‘, Journal for the American Society for Mass Spectrometry, 23 (11), (pp. 1921–1930)