Hemoglobin Raleigh in Southern Italy: a characterization with a pseudo MS3 approach of a post translational modified hemoglobin

During a routine screening for thalassemia conducted at Annunziata Hospital (Cosenza, italy), a haemoglobin variant was detected, using ion-exchange HPLC. The variant elution time was superimposable to HbA1c and it was present at 50%. For hemoglobin characterization we utilized a MALDI-TOF approach with ISD fragmentation. The spectrum of the crude hemolysate reveals the presence both of a normal ?-chain (15867 Da) and of an abnormal ?-chain (15881 Da) with a difference, between them, of +14 Da(m/z). Moreover the chromatographic characteristic of the variant were not compatible with the possibles aminoacidic change giving the +14Da difference observed . Then a MALDI-ISD analysis of the hemolysate was performed and the (same) spectra showed both the normal and a +14 abnormal c-ions pattern. The z+2 ion pattern on the contrary was normal. This results clearly indicated that the substitution concerned the N-terminal portion of the b chain. In particular because the abnormal C-ions were present starting from the ISD smaller fragment useful for analysis (C-7) (VHLTPEE), the aminoacid substitution likely was present in this fragment. A PSD analysis of the ISD abnormal fragment at m/z 967.06 (953.08+14) clearly showed on the basis of the y and b ions, that the substitution was (?1Val?Ac-Ala). This data were also confirmed by classical beta chain characterization using tryptic digestion of the abnormal globin and MS/MS analysis and by DNA sequencing. In conclusion the results obtained showed that the MALDI-ISD approach represents a rapid and effective methods for characterization of aminoacidic substitution and post-traslational modifications of Hemoglobins.