Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR - PubMed (original) (raw)

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

Sang-Won Lee et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by "mass locking" with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational 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.

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Figures

Figure 1

Figure 1

(A) Total ion current chromatogram reconstructed from FTICR mass spectra acquired during a whole capillary RPLC/FTICR experiment. (B) A 2D display of proteins in spectra 460–620. The size of the black solid circle is scaled to the intensity of a protein ion signal. Neutral masses were obtained as described in Methods. The designators AB and A/B indicate protein isoforms with identical mass and ones with different masses that eluted simultaneously, respectively. (C) Mass spectrum of proteins from spectrum number 609. The major peaks correspond to different charge states of acetylated rpL14B. rpL14B coeluted with rpL31A and rpL31B, which are denoted by ●. * indicates γ-endorphin internal standard. (Inset) An expanded mass spectrum of +15 charge state of acetylated rpL14B.

Figure 2

Figure 2

A 2D display of proteins observed in spectra 680–850 during the capillary RPLC/FTICR analysis of the yeast large ribosomal subunit (see Fig. 1 and text).

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