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. Author manuscript; available in PMC: 2012 Apr 23.
Published in final edited form as: Mol Cell. 2011 Aug 5;43(3):406–417. doi: 10.1016/j.molcel.2011.05.031

Figure 1.

Figure 1

Identification of the protein interaction network of human cyclins by immunoprecipitation and quantitative mass spectrometry. A) Schematic of the experimental strategy. HeLa cells expressing tagged cyclins were metabolically labeled with three different combinations of isotopic lysine and arginine, synchronized and immunoprecipitated with control or anti-FLAG antibodies. Immunoprecipitates were pooled and analyzed simultaneously by mass spectrometry. Peptides arising from each cell population were quantified by measuring the relative intensity of light (K0/R0), intermediate (K4/R6) and heavy (K8/R10) peaks with MaxQuant. Example schema is for cyclin A: intermediate-labeled S phase cells, and heavy-labeled cells G2 phase. For cyclin E, intermediate-labeled cells were G1 phase and heavy-labeled cells were S phase. For cyclin B, intermediate-labeled cells were G2 phase and heavy-labeled cells were M phase. B) Cyclin E complexes were isolated from G1 and S phase cells and analysed by mass spectrometry. Identified proteins are shown. Solid colored lines indicate the cut-off score for the ratio of FLAG to control IP for either G1 phase or S phase (M/L: medium/light cell populations; H/L: heavy/light populations). Each point indicates one protein identified and its color reflects the average S/G1 phase ratio (the heavy/medium ratio) of the replicate experiments. Grey points indicate the identified protein was nonspecific (not enriched above the control IP.) Purple points indicate the protein was identified in both phases, yellow, red, blue, or green shading indicates the interaction was enriched in G1, S, G2, or M phase, respectively. Note that ratios could not be calculated for all interactors as very specific interactors appear solely as heavy or medium-weight peaks with no light peak from the control sample. C) Cyclin A complexes were isolated from S and G2 phase cells. D) Cyclin B complexes were isolated from G2 and M phase cells. For complete protein identification and quantification see Supplemental Tables 1 & 5.