Matrix Effect on In-Source Decay Products of Peptides in Matrix-Assisted Laser Desorption/Ionization

摘要

MALDI-ISD of peptides were studied using several salicylic acid derivatives, 2,5-dihydroxybenzoic acid (2,5-DHB), 5-aminosalicylic acid (5-ASA), 5-formylsalicylic acid (5-FSA), and 5-nitrosalicylic acid (5-NSA) as matrices. The difference in the nature of the functional group at the 5-position in the salicylic acid derivatives can dramatically affect the ISD products. The use of 2,5-DHB and 5-ASA leads to “hydrogen-abundant” peptide radicals and subsequent radical-induced N–Cα bonds cleavage. N–Cα bond cleavage gave a c′/z^· fragment pair and radical z^·-series fragments gain a hydrogen radical or react with a matrix radical. In contrast, the use of 5-NSA resulted in the production of a “hydrogen-deficient” peptide radical that contained a radical site on the amide nitrogen in the peptide backbone. Subsequently, the radical site on the amide nitrogen induces Cα–C bond dissociation, leading to a^·/x fragment pair. The a^·-series ions undergo further hydrogen abstraction to form a-series ions after Cα–C bond cleavage. Since the Pro residue does not contain a nitrogen-centered radical site, Cα–C bond cleavage does not occur. Alternatively, the specific cleavage of CO–N bonds leads to a b^·/y fragment pair at Xxx–Pro which occurs via hydrogen abstraction from the Cα–H in the Pro residue. The use of 5-FSA generated both a^·/x- and c′/z^·-series fragment pairs. An oxidizing matrix provides useful complementary information in MALDI-ISD compared to a reducing matrix for the analysis of amino acid sequencing and site localization in cases of phosphopeptides. MALDI-ISD, when used in conjunction with both reducing and oxidizing matrices is a potentially useful method for de novo peptide sequencing.