Prompt fragmentation of disulfide-linked peptides during matrix-assisted laser desorption ionization mass spectrometry.
During the analysis of an Asp-N digest of a recombinant hematopoietic growth factor by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we observed pseudomolecular ions corresponding to reduced forms of peptides known to be present only in single disulfide linkages. Chromatographic fractionation of the peptide digest, followed by MALDI-MS and electrospray ionization (ESI) MS, confirmed that the reduced peptides were not present in the map. Fragmentation of the disulfide-linked peptides into their reduced forms occurred upon ionization from different matrices (alpha-cyano-4-hydroxycinnamic acid,2,5-dihydroxybenzoic acid, and in some instances sinapinic acid) but only after increasing the laser fluence to above threshold. Analysis of the disulfide-linked peptide fractions by ESI-MS, before and after mixing and drying with matrix, indicated that the matrix did not cause reduction. In a low-energy tandem mass spectrometric experiment with one of the cystinyl peptides, fragmentation did not occur preferentially at the disulfide bond. The pseudomolecular ions exhibited the same m/z values by MALDI-MS as their chemically reduced counterparts, indicating that they arose due to prompt fragmentation or "in-source decay" rather than "post-source decay". This finding is important for MALDI-MS analysis of peptide maps of proteins and peptide fractions with intact disulfides.
Patterson
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SD
Amgen, Inc., Amgen Center, Thousand Oaks, California 91320-1789.
Katta
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D000595:Amino Acid Sequence; D054407:Chemokine CCL4; D002851:Chromatography, High Pressure Liquid; D016207:Cytokines / Q000032:analysis* / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D019402:Macrophage Inflammatory Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015846:Monokines / Q000032:analysis* / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry
D054407:Chemokine CCL4; D016207:Cytokines; D004220:Disulfides; D019402:Macrophage Inflammatory Proteins; D015846:Monokines; D010446:Peptide Fragments; D011994:Recombinant Proteins