Oxidative radical driven cleavage of peptide backbone caused by matrix-assisted laser desorption/ionization-in-source decay with low matrix-to-peptide molar ratios

Abstract

The influence of matrix-to-analyte molar ratio (M/A) on the formation of in-source decay (ISD) fragment ions was examined by matrix-assisted laser desorption/ionization (MALDI) of peptides with the hydrogen-donating matrix 5-amino-1-naphthol (5,1-ANL). MALDI-ISD performed at a low M/A of 6.3:1 resulted in unexpected backbone cleavages that formed (a+16) and y-ions, while ISD spectra at M/A of 630:1 gave the usual fragments of c-, z’-, z-ANL and w-ions. Although the (a+16) and y-ions observed at lower M/A values could not be rationalized by conventional ISD reactions, they were in agreement with the radiolytic products caused by the oxidation of peptides with hydroxyl radical HO. MALDI mass spectra obtained at low M/A showed oxidized analyte ions [M+nO]+ (n=1–4). In addition, the spectra acquired in the low mass range showed peaks corresponding to H±, H3O±, H2O+ (or NH4+), HO−, O2− and HO2−. These results suggest that MALDI-ISD experiments performed at lower M/A values can generate active radical species such as HO, H and HO2, which leads to the oxidative radical driven cleavage of peptides.