Didia Coelho Graça1, Ralf Hartmer2, Wolfgang Jabs2, Photis Beris3,4, Lorella Clerici3, Carsten Stoermer2, Kaveh Samii3, Denis Hochstrasser1,3, Yury O. Tsybin5, Alexander Scherl1,3, Pierre Lescuyer1,3
1 University of Geneva, 2 Bruker Daltonics, 3 Geneva University Hospitals, 4 Laboratoire Unilabs Coppet, 5 Ecole Polytechnique Fédérale de Lausanne
Low and high-resolution top-down mass spectrometry (MS) methods were developed for hemoglobin disorders characterization. An automated workflow using an ion-trap with ETD capabilities allowed to identify the most clinically significant hemoglobin variants and to quantify hemoglobin chains. Analytical performances achieved with this method were compared to gold standard assays used in clinical labs. A high-resolution method was also developed for hemoglobin variants identification. Selected diagnostic product ions were used for fast and reliable data interpretation by non-expert users. MS brings more precise information at the protein level than protein analysis methods currently used for hemoglobin disorders diagnosis.
Hemoglobin (Hb) is a tetrameric blood protein that transports oxygen to organs and tissues. A healthy person carries 97% of HbA (á2β2), between 2,5% and 3.5% of HbA2 (á2ä2) and less than 1% of HbF (á2ã2). Hemoglobin disorders also called hemoglobinopathies are divided in two categories. First, qualitative disorders where a Hb variant is present due to a mutation in the protein coding sequence. Second, quantitative disorders, termed Thalassemias, result from an unbalance in the hemoglobin chains synthesis. Hb disorders diagnosis is based on the combination of clinical information, hematological tests, protein analysis techniques and DNA analysis. The protein analysis step should accurately characterize the phenotype to guide correctly DNA analysis. Nowadays, protein analysis is accomplished using a combination of several techniques such as cation-exchange liquid chromatography (CE-HPLC), gel electrophoresis and/or capillary electrophoresis. These techniques must identify the most clinically significant Hb variants, such as HbS, HbC or HbE, and accurately quantify HbA2 and HbF. HbS and HbF are also quantified for therapeutic follow-up. Nowadays, only CE-HPLC and capillary electrophoresis can perform with enough precision the quantification goal. However, due to co-elutions and co-migrations, these two techniques must be complemented by other methods to better characterize patient phenotype. In fact, the identification of rare or complex Hb variants, such as fusion proteins, can become a hard task to accomplish.
We first developed a low-resolution top-down mass spectrometry (MS) method to quickly identify the most clinically significant Hb variant and to quantify of HbA2, HbF and HbS. Variant identification is based on a top-down selected reaction monitoring (SRM) assay with electron transfer dissociation (ETD) activation in an ion-trap MS analyzer. Specific transitions were selected for HbA, HbS, HbC, HbE, HbF. For the quantification of HbA2, we developed a method based on the quantification of the isolated precursor ion without activation. The precision achieved with this strategy was below 4% of CV. All the process is automated and compatible with the clinical lab practice. A comparative study with the standard protein analysis workflow used at the Geneva University Hospitals on a cohort of 150 patient samples is ongoing.
We also developed a high-resolution top-down ETD MS method using a Q-TOF to identify mutated Hb chains. To allow data interpretation by non-expert users from the clinical area, reference product ions distributed all along the HbA β chain were selected based on their abundance, resolution and reproducibility. These diagnostic ions were associated with a color code strategy allowing to quickly and specifically localize a mutation in the Hb chain sequence. Several uncommon Hb variants including variants with only 1 Da mass shift, such as Hb G-Siriraj, and a Aã-β fusion protein named Hb Kenya were analyzed. Importantly, this method brings more precise information at the protein level compared to protein analysis methods currently used for hemoglobin disorders diagnosis. Diagnostic ion lists for á, ä and ã chains will be determined. Automation of diagnostic ions and color code strategy will also be done.
The combination of the low and high-resolution approaches allows building a complete MS platform that allows precise and efficient protein analysis in the Hb disorders diagnosis process.