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1.  Multiplexed MS/MS for Improved Data Independent Acquisition 
Nature methods  2013;10(8):10.1038/nmeth.2528.
In mass spectrometry based proteomics, data-independent acquisition (DIA) strategies have the ability to acquire a single dataset useful for identification and quantification of detectable peptides in a complex mixture. Despite this, DIA is often overlooked due to noisier data resulting from a typical five to ten fold reduction in precursor selectivity compared to data dependent acquisition or selected reaction monitoring. We demonstrate a multiplexing technique which improves precursor selectivity five-fold.
PMCID: PMC3881977  PMID: 23793237
Data Independent Acquisition; Q-Exactive; Multiplexing; Targeted Proteomics; Shotgun Proteomics
2.  Mitochondrial Respiration - An Important Therapeutic Target in Melanoma 
PLoS ONE  2012;7(8):e40690.
The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma.
PMCID: PMC3422349  PMID: 22912665
3.  Mechanisms for an effect of acetylcysteine on renal function after exposure to radio-graphic contrast material: study protocol 
Contrast-induced nephropathy is a common complication of contrast administration in patients with chronic kidney disease and diabetes. Its pathophysiology is not well understood; similarly the role of intravenous or oral acetylcysteine is unclear. Randomized controlled trials to date have been conducted without detailed knowledge of the effect of acetylcysteine on renal function. We are conducting a detailed mechanistic study of acetylcysteine on normal and impaired kidneys, both with and without contrast. This information would guide the choice of dose, route, and appropriate outcome measure for future clinical trials in patients with chronic kidney disease.
We designed a 4-part study. We have set up randomised controlled cross-over studies to assess the effect of intravenous (50 mg/kg/hr for 2 hrs before contrast exposure, then 20 mg/kg/hr for 5 hrs) or oral acetylcysteine (1200 mg twice daily for 2 days, starting the day before contrast exposure) on renal function in normal and diseased kidneys, and normal kidneys exposed to contrast. We have also set up a parallel-group randomized controlled trial to assess the effect of intravenous or oral acetylcysteine on patients with chronic kidney disease stage III undergoing elective coronary angiography. The primary outcome is change in renal blood flow; secondary outcomes include change in glomerular filtration rate, tubular function, urinary proteins, and oxidative balance.
Contrast-induced nephropathy represents a significant source of hospital morbidity and mortality. Over the last ten years, acetylcysteine has been administered prior to contrast to reduce the risk of contrast-induced nephropathy. Randomized controlled trials, however, have not reliably demonstrated renoprotection; a recent large randomized controlled trial assessing a dose of oral acetylcysteine selected without mechanistic insight did not reduce the incidence of contrast-induced nephropathy. Our study should reveal the mechanism of effect of acetylcysteine on renal function and identify an appropriate route for future dose response studies and in time randomized controlled trials.
Trial registration
Clinical NCT00558142; EudraCT: 2006-003509-18.
PMCID: PMC3293780  PMID: 22305183
Contrast-induced nephropathy; acetylcysteine; prevention; kidney; contrast media
4.  Identification of the Homeobox Protein Prx1 (MHox, Prrx-1) as a Regulator of Osterix Expression and Mediator of Tumor Necrosis Factor α Action in Osteoblast Differentiation 
Tumor necrosis factor α (TNF-α) promotes bone loss and inhibits bone formation. Osterix (Osx, SP7) is a transcription factor required for osteoblast (OB) differentiation because deletion results in a cartilaginous skeleton. We previously described a TNF suppressor element in the Osx promoter that was used to isolate nuclear proteins mediating TNF inhibition of OB differentiation. Nuclear extracts from TNF-treated pre-OBs were incubated with the TNF suppressor element for protein pull-down, and tryptic fragments were analyzed by mass spectrometry. Chromatin immunoprecipitation (ChIP) assay confirmed eight bound transcription factors. One protein, the paired related homeobox protein (Prx1), had been shown previously to have a critical role in limb bud formation and skeletal patterning. PCR revealed Prx1 expression in primary stromal cells (MSCs), C3H10T1/2 cells, and MC3T3 preosteoblasts. TNF stimulated a 14-fold increase in mRNA for Prx1, rapid cell accumulation in MC3T3 cells, and expression in periosteal and trabecular lining cells in vivo. Transient expression of Prx inhibited transcription of Osx and RUNX2. Expression of the Prx1b isoform or Prx2 decreased Osx and RUNX2 mRNA and OB differentiation in preosteoblasts. Silencing of Prx1 with siRNA abrogated TNF suppression of Osx mRNA and increased basal Osx expression. Electrophoretic mobility shift revealed Prx1b as the preferred isoform binding the Osx promoter. These results identify the homeobox protein Prx1 as an obligate mediator of TNF inhibition of Osx and differentiation of OB progenitors. Activation of Prx1 by TNF may contribute to reduced bone formation in inflammatory arthritis, menopause, and aging. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179318  PMID: 20683885
5.  Targeted Strategy for Selective Identification of Secreted Breast Tumor Proteins in Plasma Using Mouse Xenograft Models 
Early detection of breast cancer is associated with improved patient survival. While early disease is commonly identified by patient self-examination and breast mammography, interpretation of these findings are highly subjective and often require significant disease burden to achieve sensitivity. Cancer screening utilizing blood-based assays, such as measurement of prostate-specific antigen (PSA) abundance for prostate cancer, has proven to be a minimally invasive method that aids in detecting early disease. The generation of a blood-based assay for the detection of early disease in breast cancer would enable more facile disease diagnosis and thus expedite patient care.
The discovery of proteins actively shed or secreted by tumor cells into blood plasma by global proteomic analyses has proven analytically challenging, due mainly to the large dynamic range of protein abundances in blood. Common methods to enrich for tumor-specific proteins include depletion of abundant proteins from plasma samples, such as albumin and immunoglobulins. Furthermore, strategies are needed to detect blood-based candidates derived specifically from tumor cell populations to provide high-confidence candidates for further validation efforts.
To this end, we have developed a method combining global proteomic analyses of plasma collected from a mouse xenograft model of primary human breast cancer with post-data acquisition filtering of species-specific peptide search results. Primary xenograft models enable analyses of human tumor tissue in non-native biological backgrounds. Therefore, species-specific protein and gene sequences can be exploited in discovery efforts to selectively identify tumor cell-specific characteristics. Preliminary studies of plasma analyzed from xenograft-bearing mice have resulted in the identification of human-specific peptides corresponding to proteins previously described as being secreted from breast tissue and associated with breast cancer pathogenesis. Application of this strategy to proteomic analyses from a cohort of xenograft mice bearing HER2+ and triple negative breast cancer tissues will be presented.
PMCID: PMC3630706
6.  Large-Scale Automated Analysis of Location Patterns in Randomly-Tagged 3T3 Cells 
Annals of biomedical engineering  2007;35(6):1081-1087.
Location proteomics is concerned with the systematic analysis of the subcellular location of proteins. In order to perform high-resolution, high-throughput analysis of all protein location patterns, automated methods are needed. Here we describe the use of such methods on a large collection of images obtained by automated microscopy to perform high-throughput analysis of endogenous proteins randomly-tagged with a fluorescent protein in NIH 3T3 cells. Cluster analysis was performed to identify the statistically significant location patterns in these images. This allowed us to assign a location pattern to each tagged protein without specifying what patterns are possible. To choose the best feature set for this clustering, we have used a novel method that determines which features do not artificially discriminate between control wells on different plates and uses Stepwise Discriminant Analysis (SDA) to determine which features do discriminate as much as possible among the randomly-tagged wells. Combining this feature set with consensus clustering methods resulted in 35 clusters among the first 188 clones we obtained. This approach represents a powerful automated solution to the problem of identifying subcellular locations on a proteome-wide basis for many different cell types.
PMCID: PMC2901537  PMID: 17285363
Protein Subcellular Location; Subcellular Location Trees; Subcellular Location Features; CD-tagging; Fluorescence Microscopy; Cluster Analysis; Location Proteomics
Experimental cell research  2009;315(8):1415-1428.
Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCα in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCα or PKCδ in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCα and δ levels were generally reduced in colon carcinoma cell lines, PKCβII was elevated and PKCε showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCα downregulated cyclin D1 by two independent mechanisms. PKCα expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCδ had modest and variable effects on cyclin D1 steady state levels and failed to restore responsiveness to PKC agonists. Notably, PKCα expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCδ had only minor effects. Loss of PKCα and effects of its re-expression were independent of the status of the APC/β-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCα is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the frequent loss of this isozyme during intestinal carcinogenesis.
PMCID: PMC2721478  PMID: 19232344
Protein Kinase C; Cyclin D1; Transcriptional Control; Colon Cancer; 4E-BP1; β-Catenin; Mouse Models

Results 1-7 (7)