Abstract
The characterization of heterogeneous multicomponent protein complexes, which goes beyond identification of protein subunits, is a challenging task. Here we describe and apply a comprehensive method that combines a mild affinity purification procedure with a multiplexed mass spectrometry approach for the in-depth characterization of the exosome complex from Saccharomyces cerevisiae expressed at physiologically relevant levels. The exosome is an ensemble of primarily 3' --> 5' exoribonucleases and plays a major role in RNA metabolism. The complex has been reported to consist of 11 proteins in molecular mass ranging from 20 to 120 kDa. By using native macromolecular mass spectrometry we measured accurate masses (around 400 kDa) of several (sub)exosome complexes. Combination of these data with proteolytic peptide LC tandem mass spectrometry using a linear ion trap coupled to a FT-ICR mass spectrometer and intact protein LC mass spectrometry provided us with the identity of the different exosome components and (sub)complexes, including the subunit stoichiometry. We hypothesize that the observed complexes provide information about strongly and weakly interacting exosome-associated proteins. In our analysis we also identified for the first time phosphorylation sites in seven different exosome subunits. The phosphorylation site in the Rrp4 subunit is fully conserved in the human homologue of Rrp4, which is the only previously reported phosphorylation site in any of the human exosome proteins. The described multiplexed mass spectrometry-based procedure is generic and thus applicable to many different types of cellular molecular machineries even if they are expressed at endogenous levels.
Highlights
The characterization of heterogeneous multicomponent protein complexes, which goes beyond identification of protein subunits, is a challenging task
Affinity purification coupled to mass spectrometry has proven to be a very powerful approach (9, 10); it allows the analysis of protein complexes, which are expressed at physiological levels from endogenous promoters and are assembled in vivo (3, 6, 11)
We report on the use of macromolecular MS to characterize the genuine stronger physical interactions and subunit stoichiometry of the exosome complex from Saccharomyces cerevisiae, which was expressed at endogenous levels and purified using the tandem affinity purification (TAP) procedure (9, 10)
Summary
S. cerevisiae Strain, Cultivation, and Protein Purification—The S. cerevisiae strain MGD35313D, BSY17 containing Csl or Rrp as the C-terminal tagged entry point was kindly provided by Cellzome AG (Heidelberg, Germany) (3). 2 liters of cell culture of S. cerevisiae was grown at 30 °C in yeast extract-peptone-dextrose medium to an optimal density at 600 nm of 3.8. After reducing the flow to 100 nl/min by using a splitter, the peptides were transferred to the analytical column (Reprosil C18AQ; 200 mm ϫ 50 m, packed in house) with a linear gradient from 0 to 50% eluent B (0.1 M acetic acid in 80% (v/v) acetonitrile) for 60 min. After reducing the flow to 1 l/min by using a splitter, the proteins were transferred to the analytical column (Vydac TP214 C4RP; 123 mm ϫ 150 m, 5-m particle size, packed in house) with a linear gradient from 0 to 80% eluent B (0.05% (v/v) trifluoroacetic acid, 80%. Exosome sample (1 pmol; concentration, 0.5 M) was introduced into the modified electrospray ionization time-of-flight instrument mass spectrometer using nanoflow electrospray glass capillaries. The mass spectra were externally calibrated with 40 mg/ml cesium iodide and analyzed by MassLynx 4.0 software (Waters)
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