PNNL

Abstract

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry coupled with capillary reverse phase LC (RPLC) was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accurancy, achieved by "mass locking" with an internal standard froma dual ESI source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on co-translational and post-translational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.