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–C
α bonds cleavage. N–C
α 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 C
α–C bond dissociation, leading to
a·/
x fragment pair. The
a·-series ions undergo further hydrogen abstraction to form
a-series ions after C
α–C bond cleavage. Since the Pro residue does not contain a nitrogen-centered radical site, C
α–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 C
α–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.
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