All constructs were verified by automated DNA sequencing

a similar number of proteins as had been identified with MaxQuant. Both search strategies generated overlapping protein lists. Once results 12149260 were gathered from both programs, the results were combined. When proteins were identified by both programs, the quantification calculated by the MaxQuant software was reported. If the ratios were such that one program defined a protein as changed whereas the second program did not, the ratios were manually calculated through integration of the peak areas using the XCalibur software. Proteins were divided into subsets based on their SILAC ratios using a 1.5-fold change as the cutoff threshold. That is, a ratio of 1.5 or higher was scored as an increase whereas a ratio of 0.666 or less was scored as a decrease; ratios that fell between these values were reported as no change. These ratios, as well as the log2 transformations, are reported in Results Synchronous HeLa Cells Progressing 23446639 through the G1/S and S/G2 Transitions We sought to investigate the 606143-89-9 web proteome changes between G1 and S phase and between S and G2 phase. Our goal was to achieve very tight cell cycle synchrony while simultaneously avoiding strong checkpoint effects that could be induced in chemicallyarrested cells. To facilitate accurate quantification of peptides by mass spectrometry, we labeled cultures for more than 5 cell divisions with three different stable isotope mixtures of lysine and arginine prior to synchronization. To obtain populations of isotope-labeled tightly-synchronous cells progressing from G1 to S phase, we modified the Whitfield et al. double-thymidine block and release protocol . We released HeLa cells from the second thymidine block to allow checkpoint recovery and normal passage through the subsequent transitions and allowed them to progress into mitosis without further chemical perturbation. We collected mitotic cells using a “shake-off”method, a procedure that takes advantage of the tenuous attachment of HeLa cells as they round up during mitosis. We replated mitotic cells in fresh dishes, and 3 hrs after mitosis, the cells were a relatively pure population of G1 cells; by 10 hrs after mitosis they were in early-S phase. Note that these cell cycle times reflect a moderate delay compared to cells grown under standard conditions due to the requirement for dialyzed fetal bovine serum for efficient metabolic labeling. To facilitate the detection of proteins that may be rapidly degraded in S phase we treated another culture of cells with the proteasome inhibitor MG132 8 hrs after the mitotic shake-off and harvested the cells 2 hrs later in early S phase. To quantify proteins that change between S phase and G2 phase, we released cells into S phase from the doublethymidine block rather than from a mitotic shake-off. These cells progressed through S phase and entered G2 phase synchronously; we harvested 3 hrs and 8 hrs after release from the second thymidine block. We also treated cells with MG132 6 hrs after release and harvested them 2 hrs later. For the G1/S comparison, the G1 culture contained normal isotopes, the early-S phase culture was metabolically labeled with intermediate isotopes, and the early-S phase culture treated with MG132 at the G1/S transition had been cultured in the heaviest isotopes. For the S/G2 comparison, mid-S phase cells were cultured in the normal isotope medium, the G2 cells were cultured in the intermediate isotope medium, and the G2 cells that had been treated with MG132 at the S/G2 transi