Haemoglobin variant identification using the Thermo Fisher TSQ Quantum Ultra

Currently, there are over 1160 haemoglobin (Hb) variants registered on the Globin Gene Server database.1 Variant haemoglobins are usually the result of an amino acid substitution in the ‘normal’ alpha- or beta-globin chain. The function of the Hb molecule can be affected when a mutation arises to produce a variant Hb, and in some cases this can cause significant clinical implications for the individual carrying this mutation. Current screening techniques are able to identify the presence of an abnormal Hb but are unable to give a positive identification to that variant, and a secondary method is required for this purpose.

In 2001 Wild et al.2 described a method for the rapid identification of Hb variants by electrospray ionisation mass spectrometry (ESI-MS) using the Waters Quatro II mass spectrometer. The basis of this method can be separated into four steps: i) identification of an abnormal Hb variant by a primary high-performance liquid chromatography (HPLC) method and the preparation of the patient sample for ESI-MS; ii) analysis and deconvolution of the intact masses of the alpha- and beta-globin chains to detect any mass change created by the Hb variant, and therefore deduce a list of possible variants responsible for the mass change; iii) tryptic digestion of the sample and analysis by MS to identify in which tryptic fragment the variant was located; and iv) ESI-MS/MS on the variant tryptic fragment to identify the mutant amino acid responsible for the Hb variant.

The aim of the current study is to utilise the principles of this method to develop an application for the Thermo Fisher TSQ Quantum Ultra mass spectrometer and its software with the addition of a liquid chromatography step to identify the retention times of the normal and variant tryptic fragments.