Characterization of N-terminal acetylated Hb Raleigh by a MALDI-ISD approach

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 HbAlc and it was present at 50%. For Haemoglobin characterization we utilized a MALDI-TOF approach with ISD fragmentation. The spectrum of the crude hemolysate reveals the presence both of a normai beta-chain (MW 15867, 1) and of an abnormal beta-chain (MW 15881,3) with a difference, between them, of +14 Da. Moreover, the chromatographic features of the variant were not compatible with the possible aminoacidi e change giving the + 14 Da difference observed. Then a MALDI-ISD analysis of the hemolysate was performed and the same spectra showed both the normai and the + 14 Da abnormal c-ions pattern. The (z+2)-ion pattern on the contrary was normai. This results clearly indicated that the substitution concerned the N-terminal portion of the beta chain. Because the abnormal c-ions were present starting from the ISD smaller fragment useful for analysis betac7 (VHL TPEE), the amino acid substitution likely was present in this fragment. A PSD analysis ofthe ISD abnormal fragment at m/z 967.06 (953.08+14) clearly showed, based on the y- and b-ions, that the substitution was (b1 Val7 Ac-Ala). This data was also confirmed by classica] 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 top-down MALDI-ISD approach represents a rapid and effective methods for characterization of aminoacidic substitution and post-translational modifications ofh emoglobins