Abstract
Dynamically interacting proteins associate and dissociate with their binding partners at high on/off rates. Although their identification is of great significance to proteomics research, lack of an efficient strategy to distinguish stable and dynamic interactors has hampered the efforts toward this goal. In this work, we developed a new method, MAP (mixing after purification)-SILAC (stable isotope labeling of amino acids in cell culture), to quantitatively investigate the interactions of protein complexes by mass spectrometry. In combination with the original SILAC approach, stable and dynamic components were effectively distinguished by the differences in their relative abundance ratio changes. We applied the newly developed strategies to decipher the dynamics of the human 26 S proteasome-interacting proteins. A total of 67 putative human proteasome-interacting proteins were identified by the MAP-SILAC method among which 14 proteins would have been misidentified as background proteins due to low relative abundance ratios in standard SILAC experiments and 57 proteins have not been reported previously. In addition, 35 of the 67 proteins were classified as stable interactors of the proteasome complex, whereas 16 of them were identified as dynamic interactors. The methods reported here provide a valuable expansion of proteomics technologies for identification of important but previously unidentifiable interacting proteins.
Highlights
Interacting proteins associate and dissociate with their binding partners at high on/off rates
Quantifying Dynamic Interactors of Protein Complexes light and heavy labeled forms during the purification, the fast on/off rates of dynamically interacting proteins will result in an equilibrium between the two forms of the proteins that are bound to the bait
In this work we report a new method for quantitative identification of dynamic interactors of protein complexes
Summary
Chemicals and Reagents—ImmunoPure streptavidin, HRP-conjugated antibody, and Super Signal West Pico chemiluminescent substrate were from Pierce. If peptides matched to multiple members of a protein family, criteria used in the Search Compare program for selecting the ones to report are as follows. For the SILAC experiments, the Search Compare program was used to calculate the relative abundance ratios of Arg/Lys-containing peptides based on ion intensities of monoisotopic peaks observed in the LC MS spectra at the time when the peptides were sequenced and subsequently identified during database searching [7, 21]. The SILAC ratios were further validated by checking all of the raw spectra within the Protein Prospector Search Compare program. In MAP experiments, equal amounts of lysates A and B were subjected to purification separately by incubating with streptavidin resin for 2 h. ADRM1-FLAG protein was detected using a mouse anti-FLAG antibody (1:2000) followed by HRP-conjugated anti-mouse IgG (1:10,000). The purified complexes were digested with trypsin and analyzed by LC MS/MS as described above
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