Detection, number, and sequence location of sulfur-containing amino acids and disulfide bridges in peptides by ultrahigh-resolution MALDI FTICR mass spectrometry.

Here, we present several strategies for determining the number of sulfur atoms and disulfide bridges in selected biologically active peptides, based on MALDI FTICR mass spectrometry at femtomole sample consumption level. First, based on the 2-Da mass increase per disulfide bridge reduction, we show that repeated laser shots on the same sample spot can reduce (and therefore reveal the presence of) the disulfide bridge in oxytocin. Second, we show that the primary sequence positions of the disulfide-bridged cystines can be inferred from the presence/absence of MALDI-induced reduction in cystine-containing fragment ions. Third, we show that the presence and number of sulfur atoms as well as the degree of reduction in a peptide can all be determined directly from isotopic relative abundances of mass-resolved 34S, 13C2, and reduced all-12C species in a single ultrahigh-resolution MALDI FTICR mass spectrum. Methods for achieving such ultrahigh mass resolution of peptide ions of closely spaced m/z (m/delta m50% approximately 950,000 at m/z approximately 650) at modest magnetic field (3 T) are discussed.