A method for simple identification of signature peptides derived from polyUb-K48 and K63 by MALDI-TOF MS and chemically assisted MS/MS fragmentation

A simple method is described to identify signature peptides derived from polyubiquitin (polyUb) chains. The method is based on MALDI-TOF MS/MS analysis after chemically assisted fragmentation, and works on peptides isolated from polyacrylamide gels. PolyUb chains branched at K48 and K63 were chosen as models for Ub-protein conjugates. They were resolved by SDS-PAGE, and their tryptic peptides (in-gel-trypsinolysis) derivatized with 3-sulfopropinic acid NHSester to obtain chemically assisted fragmentation during the MS/MS analysis. PolyUb-K63 produced a single peptide identified as 55TLSDYNIQK63 (GG)ESTLHLVLR72. PolyUb-K48 produced two branched signature peptides identified as 43LIFAGK48(GG)QLEDGR54 and 43LIFAGK48(LRGG)QLEDGR54. The recovery of signature peptide with LRGG as branched chain underscores the need to take limited proteolysis into account in the search for detection of ubiquitinated peptides in proteomics studies. In conclusion, a simple method has been described allowing the identification of signature peptides, which are diagnostic markers of the majority of polyUb-conjugated proteins. In principle, the method should be applicable also for other more rare signature peptides.