Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication.

The endometrial fluid is a noninvasive sample which contains numerous secreted proteins representative of endometrial function and reflects the state of the endometrium. In this study, we describe, for the first time, a comprehensive catalogue of proteins of the endometrial fluid during the secretory phase of the menstrual cycle. To achieve this objective, three different but complementary strategies were used: First, in-solution digestion followed by reverse phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS); second, protein separation by denaturing one-dimensional electrophoresis (SDS-PAGE) followed by HPLC-MS/MS analysis. Finally, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by MALDI-TOF/TOF analysis. The combination of the three strategies led to the successful identification of 803 different proteins in the International Protein Index (IPI) human database (v3.48). An extensive description of the endometrial fluid proteome will help provide the basis for a better understanding of a number of diseases and processes, including endometriosis, endometrial cancer and embryo implantation. We believe that the thorough catalogue of proteins presented here can serve as a valuable reference for the study of embryo implantation and for future biomarker discovery involved in pathologic alterations of endometrial function.

Quantitation of changes in protein abundance is key to understanding the alterations that biological systems undergo and to discovering novel biomarkers. Currently, HPLC‐MS/MS can be used to quantify changes in protein expression levels [Ong, S. E. and Mann, M., Nat. Chem. Biol. 2005, 1, 252–262]. Nevertheless, quantitative analysis of protein mixtures by HPLC‐MS/MS is still hampered by the wide range of protein expression levels, the high dynamic range of protein concentrations and the lack of reliable quantitation algorithms [D'Ascenzo, M., et al. Brief. Funct. Genomic. Proteomic. 2008, 7, 127–135; Lin, W. T., et al., J. Proteome Res. 2006, 5, 2328–2338; Matthiesen, R., et al. J. Proteome Res. 2005, 4, 2338–2347; Yu, C. Y., et al. Nucleic Acids Res. 2007, 35, W707–W712]. In this context, we describe two different samples (4‐protmix and 8‐protmix) suitable for relative protein quantitation using iTRAQ. Using the 4‐protmix, relative protein changes of up to 24‐fold were measured. The 8‐protmix allowed the quantitation of the relative protein changes in a mixture of proteins within the range of two orders of magnitude in concentration and ten‐fold differences in relative abundance. We propose that the two samples are suited to test the iTRAQ quantitative proteomic workflow. We analyzed the iTRAQ samples with a LTQ Orbitrap using “higher energy collision‐induced dissociation” fragmentation [Olsen, J. V., et al., Nat. Methods 2007, 4, 709–712] and quantified with Proteome Discoverer v.1.1 (Thermo Fisher Scientific). We believe that the presented protein mixtures will be useful to assess the performance of the iTRAQ‐based quantitation proteomic strategy in any laboratory.