Tandem mass spectrometric analysis of complex biological mixtures

Abstract

Proteomics is suddenly one of the hottest research areas of science, inspired by the completion of the sequencing of the human genome and of genomes of a number of other key species. The genome’s DNA sequence predicts those of its expressed proteins, and mass spectrometry (MS) is already playing a dominant role in the qualitative and quantitative analysis of these complex protein mixtures. Tandem mass spectrometry (MS/MS) for targeted compound analysis, pioneered by Cooks, is now a key technique for such problems, and this paper tries to show how it will be even more important in the future. The most used current MS approach to proteome analysis is applied after enzymatic degradation produces a complex mixture of peptides that provide MS or MS/MS sequence information. In the top down MS approach, ionization of protein mixtures yields mixtures of molecular ions that can be separated by MS for MS/MS sequence characterization of the selected “purified” protein. All posttranslational modifications and errors will be represented if MS/MS yields a complementary set of fragment ions, something not usually achieved with the smaller pieces of the bottom up approach. Here the new technique of electron capture dissociation (ECD) shows great promise, providing for more backbone cleavages than conventional methods and designating N- and C-terminal fragment ions. ECD specifically dissociates backbone bonds without appreciable loss of glycosylation, phosphorylation, or other posttranslational modifications, in contrast to energetic dissociation methods. This appears especially applicable for a proteome problem that involves quantitative determination of the extent of phosphorylation at two of 16 phosphorylation sites in the 50 kDa Lyn kinase.