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A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.

Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.The Polycomb group (PcG) genes were first identified by their roles in ensuring appropriate temporal and spatial expression of Homeotic (Hox) genes, which act as transcriptional repressors required for correct body patterning during development. Members of the PcG family also play important regulatory roles in adult organisms, including stem cell maintenance, differentiation, and malignant transformation (for a review, see references 15, 23, and 42).PcG members are generally subdivided into two groups, Polycomb repressive complex 1 (PRC1) and PRC2, and are associated with distinct multiprotein complexes (13,30). It is believed that a coordinated action between PRC1 and PRC2 complexes introduces heritable histone modifications at certain promoters, followed by the recruitment of additional transcriptional repressors to these sites. The PRC2 complex, which includes Ezh2 and EED, possesses histone H3 lysine 9 and 27 methyltransferase activity, a "silencing" histone mark (8,10,38). PRC1 components subsequently bind to methylated H3K27, inhibit SWI/SNF remodeling, and recruit histone deacetylases to silence gene expression (11,47). Biochemical histone modification by components of the PRC1 complex has also been described: reconstituted PRC1-like complexes containing Bmi1 and RING1B contain an E3 ubiquitin ligase activity capable of ubiquitinating histone H2A (7, 46).Bmi-1 and Mel-18 are highly homologous constituents of the PRC1 complex. They share 65% amino acid identity and contain a RING finger domain in the N terminus, as well as proline-rich sequences and a helix-turn-helix domain in the C-terminal tail. While they are viable and have...

Both data in preclinical cancer models and data with T cells from patients with advanced cancer support a role for Tim-3 blockade in promoting effective anti-tumor immunity. Consequently, there is considerable interest in the clinical development of antibody-based therapeutics that target Tim-3 for cancer immunotherapy. A challenge to this clinical development is the fact that several ligands for Tim-3 have been identified: galectin-9, phosphatidylserine, HMGB1, and most recently, CEACAM1. These observations raise the important question of which of these multiple receptor:ligand relationships must be blocked by an anti-Tim-3 antibody in order to achieve therapeutic efficacy. Here, we have examined the properties of anti-murine and anti-human Tim-3 antibodies that have shown functional efficacy and find that all antibodies bind to Tim-3 in a manner that interferes with Tim-3 binding to both phosphatidylserine and CEACAM1. Our data have implications for the understanding of Tim-3 biology and for the screening of anti-Tim-3 antibody candidates that will have functional properties .

Multidimensional separations combined with mass spectrometry are used to study the proteins that are present in two states of Drosophila melanogaster: the whole embryo and the adult head. The approach includes the incorporation of a gas-phase separation dimension in which ions are dispersed according to differences in their mobilities and is described as a means of providing a detailed analytical map of the proteins that are present. Overall, we find evidence for 1133 unique proteins. In total, 780 are identified in the head, and 660 are identified in the embryo. Only 307 proteins are in common to both developmental stages, indicating that there are significant differences in these proteomes. A comparison of the proteome to a database of mRNAs that are found from analysis by cDNA approaches (i.e., transcriptome) also shows little overlap. All of this information is discussed in terms of the relationship between the predicted genome, and measured transcriptomes and proteomes. Additionally, the merits and weaknesses of current technologies are assessed in some detail.