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1.  BRD4 Short Isoform Interacts with RRP1B, SIPA1 and Components of the LINC Complex at the Inner Face of the Nuclear Membrane 
PLoS ONE  2013;8(11):e80746.
Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. BRD4, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.
doi:10.1371/journal.pone.0080746
PMCID: PMC3834312  PMID: 24260471
2.  Proteomic Screening of Human Targets of Viral microRNAs Reveals Functions Associated with Immune Evasion and Angiogenesis 
PLoS Pathogens  2013;9(9):e1003584.
Kaposi's sarcoma (KS) is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). The virus expresses unique microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC) in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1) following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1╬▒), and up-regulation of a protein involved in stimulating angiogenesis (HMOX1). This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.
Author Summary
Kaposi's sarcoma-associated herpesvirus is the virus associated with multiple proliferative disorders, including Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. This virus expresses small nucleic acids (with sequences distinct from other organisms), called microRNAs, that can limit expression of specific genes. Currently, we only know a few validated targets of these viral microRNAs and the mechanisms of microRNA-mediated repression are still being actively debated. We used a method to look at protein expression changes induced by these viral microRNAs to better understand microRNA targets and functions. The method we describe here found microRNA targets that are missed by other approaches. In addition to identifying previous microRNA targets and discovering new microRNA targets, we found the function of specific viral microRNAs to be associated with immune evasion and the expansion of blood vessel networks, a hallmark of Kaposi's sarcoma. The results may be a resource for those studying microRNAs from other organisms, and furthermore, the microRNA functions described provide mechanistic insight into viral pathogenesis and immune evasion.
doi:10.1371/journal.ppat.1003584
PMCID: PMC3764211  PMID: 24039573
3.  Comparison of Strong Cation Exchange and SDS/PAGE Fractionation for Analysis of Multi-Protein Complexes 
Journal of proteome research  2010;9(12):6696-6704.
Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. While affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes pre-fractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Pre-fractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data shows that pre-fractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.
doi:10.1021/pr100843x
PMCID: PMC3707127  PMID: 20968308
strong cation exchange; immuno-precipitation; protein complex isolation; mass spectrometry; FLAG
4.  18O Stable Isotope Labeling in MS-based Proteomics 
A variety of stable isotope labeling techniques have been developed and used in mass spectrometry (MS)-based proteomics, primarily for relative quantitation of changes in protein abundances between two compared samples, but also for qualitative characterization of differentially labeled proteomes. Differential 16O/18O coding relies on the 18O exchange that takes place at the C-terminal carboxyl group of proteolytic fragments, where two 16O atoms are typically replaced by two 18O atoms by enzyme-catalyzed oxygen-exchange in the presence of H218O. The resulting mass shift between differentially labeled peptide ions permits identification, characterization and quantitation of proteins from which the peptides are proteolytically generated. This review focuses on the utility of 16O/18O labeling within the context of mass spectrometry-based proteome research. Different strategies employing 16O/18O are examined in the context of global comparative proteome profiling, targeted subcellular proteomics, analysis of post-translational modifications and biomarker discovery. Also discussed are analytical issues related to this technique, including variable 18O exchange along with advantages and disadvantages of 16O/18O labeling in comparison with other isotope-coding techniques.
doi:10.1093/bfgp/eln055
PMCID: PMC2722262  PMID: 19151093
18O labeling; enzyme-mediated isotope incorporation; stable isotope labeling; MS-based proteomics; relative protein quantitation; LC/MS/MS

Results 1-4 (4)