Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO Red staining

The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting, Proteins which are not chemically modified during the SYPR O Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-as sisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red st aining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins From a total of 31 selected protein spots, The peptide mass signal intensity, the number of matched peptides and the p ercentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modifie d silver protein gel staining. This work demonstrates that fluorescent SYPR O Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver s taining. As far as we know, this is the first report in which C. albicans p roteins separated using 2-D gels have been identified by peptide mass finge rprinting, The improved technique described here should be very useful for carrying out proteomic studies. Copyright (C) 2000 John Wiley & Sons, Ltd.