Premium
Monitoring ribo‐proteome pool sizes by isotope pulse mass spectrometry
Author(s) -
Sykes Michael Timothy,
Chen Stephen Suo,
Sperling Edit,
Williamson James Russell
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.225
Subject(s) - ribosomal protein , ribosome , proteome , mass spectrometry , chemistry , kinetics , ribosomal rna , isotope , stable isotope labeling by amino acids in cell culture , chromatography , label free quantification , biophysics , quantitative proteomics , proteomics , biology , biochemistry , rna , physics , quantum mechanics , gene
Protein pool sizes are determined by the balance of synthesis and utilization kinetics, and reflect the dynamics of protein turnover. We have developed a quantitative method to measure pool sizes using stable isotope pulse labeling and liquid chromatography coupled mass spectrometry. Quantitative analysis of proteins from ribosomes recovered at different times allows us to quantify the kinetics of incorporation of 15 N into each individual ribosomal protein. Estimated pool sizes range from 2% to 40% for proteins in the 30S subunit, though most have a pool size of approximately 10%. Proteins with unusually large pool sizes turn out to be those with multiple known functions. Pool sizes generally correlate with the in vitro assembly order, and using this relationship along with an estimate of the total assembly time of a single ribosome we are able to deconvolute the relative contributions of free proteins and ribosomal intermediates to the total pool. Our research represents a new way to monitor pool sizes and protein pool dynamics in vivo while gaining special insight into the ribo‐proteome. We expect the methods used to be generally applicable to a wide variety of cellular systems. M.T.S. supported by NIH grant F32GM083510.