• Bulletin of the Korean Chemical Society
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• v.33 no.10
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• pp.3233-3240
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• 2012

We demonstrated the combined applications of online protein digestion using trypsin immobilized enzyme reactor (IMER) and dual tandem mass spectrometry with collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) for tryptic peptides eluted through the trypsin-IMER. For the trypsin-IMER, the organic and inorganic hybrid monolithic material was used. By employing the trypsin-IMER, the long digestion time could be saved with little or no sacrifice of the digestion efficiency, which was demonstrated for standard protein samples. For three model proteins (cytochrome c, carbonic anhydrase, and bovine serum albumin), the tryptic peptides digested by the IMER were analyzed using LC-MS/MS with the dual application of CAD and ETD. As previously shown by others, the dual application of CAD and ETD increased the sequence coverage in comparison with CAD application only. In particular, ETD was very useful for the analysis of highly-protontated peptide cations, e.g., ${\geq}3+$. The combination approach provided the advantages of both trypsin-IMER and CAD/ETD dual tandem mass spectrometry applications, which are rapid digestion (i.e., 10 min), good digestion efficiency, online coupling of trypsin-IMER and liquid chromatography, and high sequence coverage.

• Mass Spectrometry Letters
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• v.1 no.1
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• pp.5-8
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• 2010

In the present study, collisionally-activated dissociation (CAD) experiments were performed under low energy collision conditions in six peptides containing a disulfide bond. Fragments produced as a result of the cleavage of a disulfide bond were obtained after CAD in four peptides (bactenecin, TGF-$\alpha$, cortistantin, and linearly linked peptide, Scheme 1) with basic amino acid residues. In contrast, the CAD analysis of two peptides with no basic residue (oxytocin and tocinoic acid) rarely produced fragments indicative of cleavage of a disulfide bond. These results are consistent with the mobile proton model suggested by the McLuckey and O'air groups (ref. 22 and 23); nonmobile protons sequestered at basic amino acid residues appear to promote the cleavage of disulfide bonds.