APPLICATIONS OF IN-SOURCE FRAGMENTATION OF PROTEIN IONS FOR DIRECT-SEQUENCE ANALYSIS BY DELAYED EXTRACTION MALDI-TOF MASS-SPECTROMETRY

In matrix-assisted laser desorption/ionization of proteins, there exis ts a certain amount of fast metastable decay immediately after laser i rradiation. The fragment ions thus formed can be resolved and their m/ z values measured accurately by employing delayed extraction linear ti me-of-night mass spectrometry. At higher than threshold laser fluences , proteins exhibit a series of fragment ions providing useful sequence information. We also observe that when moderate amounts of salts are present in the sample with sinapinic acid being the matrix, the intens ities of c(n) ions (N-terminal fragments) are enhanced compared to oth er types of fragment ions. This enhancement in c(n) ion signals allows direct sequencing of proteins. The c(n) ions are completely absent wh en Xxx-Pro bonds are encountered and are of lon;er intensity when Xxx- Gly bonds are involved. Further, the c(n) ion series is interrupted at Xxx-Cys, when the cysteine is involved in a disulfide bond. Upon redu ction of the disulfide bonds, the series continues and information is available for longer stretches. Using 10-20 pmol of recombinant protei ns, sometimes contiguous sequence information up to 70 residues is obt ained in a matter of minutes. Applications of the technique to some re combinant proteins with intra- or interchain disulfide linkages are pr esented.