Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR.
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.
Lee
Sang-Won
SW
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
Berger
Scott J
SJ
Martinović
Suzana
S
Pasa-Tolić
Ljiljana
L
Anderson
Gordon A
GA
Shen
Yufeng
Y
Zhao
Rui
R
Smith
Richard D
RD
D002845:Chromatography / Q000379:methods*; D019175:DNA Methylation; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D010766:Phosphorylation; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012984:Software; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D007477:Ions; D020033:Protein Isoforms