abundance changed from G1 to S or from S to G2 have been detected as phosphoproteins, consistent with the notion that many protein abundance changes are controlled by phosphorylation. This enrichment was true both for proteins that changed from G1 to S and for those that changed from S to G2. Since the cyclin-dependent kinases govern many cell cycle transitions, we compared our sets of regulated proteins with a list of candidate Cdk substrates. Many proteins that increased or decreased in S phase appear on this list of Cdk substrates. Moreover, a statistically significant number of proteins that increased in G2 phase are also putative Cdk substrates. A significant number of proteins that changed with MG132 treatment at the S/G2 transition are also putative Cdk substrates. In contrast, proteins that changed in response to MG132 treatment at the G1/S transition were not enriched for putative Cdk substrates. Like Cdks, the ATR kinase is active during S phase. ATR activity is also stimulated by DNA damage, and this property was used to identify candidate ATR substrates. Putative ATR kinase substrate lists were developed by Stokes 10501907 et al. from phosphopeptides detected following UV irradiation, an activator of ATR. A subset of our regulated proteins also appeared in these lists of potential ATR substrates. The majority of proteins that change with MG132 treatment,, were not ATR substrates, but proteins that decreased with MG132 treatment at the S/G2 transition were significantly enriched in ATR substrates. Taken together, these comparisons are consistent with the prevailing model that many changes in protein abundance between G1 and S phase and between S and G2 phase are associated with both protein ubiquitination and protein phosphorylation, but this analysis also underscores the idea that only some changes, particularly as cells progress from G1 to S phase in continuously growing cells, are due solely to mRNA 946128-88-7 fluctuations. Unanticipated Cell Cycle-regulated Proteins Include Alternative pre-mRNA Splicing Factors Cell Cycle-Regulated Proteome: Splicing Proteins S phase and the set that increased from S to G2 phase. Both sets of MG132-sensitive proteins were also enriched for RNA processing and ribonucleoprotein complex biogenesis proteins. The striking enrichment of pre-mRNA processing proteins in the collection of proteins that were down-regulated in S phase prompted us to analyze those proteins more directly. In particular, the enriched GO terms included nuclear 9305921 pre-mRNA splicing, and more specifically, alternative splicing. Of the 244 known splicing factors, we detected 72 core proteins and 65 noncore proteins . Additionally, we detected 58.7% of the non-core spliceosome machinery, and 62.3% of these subunits decreased in S phase. Strikingly, we quantified almost all of the known heterogeneous nuclear ribonucleoproteins, and 72.7% of these proteins decrease in S phase. These 
proteins are important in determining exon inclusion, suggesting that alternative splicing is particularly affected during S phase. We probed several of the alternative splicing factors by immunoblotting to determine if the changes observed by mass spectrometry were valid. As shown in Discussion Previous unbiased analyses of the human transcriptome and proteome have generated an appreciation for the interconnectedness of different biochemical pathways. Inspired by such findings, we considered it likely that the human cell cycle includes changes not only in the well-stu