Search tips
Search criteria

Results 1-25 (29)

Clipboard (0)

Select a Filter Below

Year of Publication
more »
Document Types
1.  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
2.  Improved Precision of Proteomic Measurements in Immunoprecipitation Based Purifications Using Relative Quantitation 
Analytical chemistry  2013;85(9):4301-4306.
Mass spectrometry coupled immunoprecipitation (MS-IP) studies are useful in identifying and quantitating potential binding partners of a target protein. However, they are often conducted without appropriate loading controls. Western blots are often used to analyze loading controls, yet there are limitations to their usefulness as analytical tools. One remedy for this is the use of selected reaction monitoring (SRM), where the areas under the curve (AUCs) of peptides from a protein of interest can be normalized to those from the constant regions of the immunoglobulins used for the IP. Using this normalization method, significant changes in relative peptide abundance were observed between samples when there appeared to be an unequal load based on immunoglobulin peptide abundance.
PMCID: PMC3995537  PMID: 23517085
3.  Context-Sensitive Markov Models for Peptide Scoring and Identification from Tandem Mass Spectrometry 
Peptide and protein identification via tandem mass spectrometry (MS/MS) lies at the heart of proteomic characterization of biological samples. Several algorithms are able to search, score, and assign peptides to large MS/MS datasets. Most popular methods, however, underutilize the intensity information available in the tandem mass spectrum due to the complex nature of the peptide fragmentation process, thus contributing to loss of potential identifications. We present a novel probabilistic scoring algorithm called Context-Sensitive Peptide Identification (CSPI) based on highly flexible Input-Output Hidden Markov Models (IO-HMM) that capture the influence of peptide physicochemical properties on their observed MS/MS spectra. We use several local and global properties of peptides and their fragment ions from literature. Comparison with two popular algorithms, Crux (re-implementation of SEQUEST) and X!Tandem, on multiple datasets of varying complexity, shows that peptide identification scores from our models are able to achieve greater discrimination between true and false peptides, identifying up to ∼25% more peptides at a False Discovery Rate (FDR) of 1%. We evaluated two alternative normalization schemes for fragment ion-intensities, a global rank-based and a local window-based. Our results indicate the importance of appropriate normalization methods for learning superior models. Further, combining our scores with Crux using a state-of-the-art procedure, Percolator, we demonstrate the utility of using scoring features from intensity-based models, identifying ∼4-8 % additional identifications over Percolator at 1% FDR. IO-HMMs offer a scalable and flexible framework with several modeling choices to learn complex patterns embedded in MS/MS data.
PMCID: PMC3567622  PMID: 23289783
4.  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
5.  Quaternary ammonium silane-functionalized, methacrylate resin composition with antimicrobial activities and self-repair potential 
Acta biomaterialia  2012;8(9):3270-3282.
Design of antimicrobial polymers for enhancing healthcare issues and minimizing environmental problems is an important endeavor with both fundamental and practical implications. Quaternary ammonium silane-functionalized methacrylate (QAMS) represents an example of antimicrobial macromonomers synthesized by a sol-gel chemical route; these compounds possess flexible Si-O-Si bonds. In present work, a partially-hydrolyzed QAMS copolymerized with bis-GMA is introduced. This methacrylate resin was shown to possess desirable mechanical properties with both a high degree of conversion and minimal polymerization shrinkage. Kill-on-contact microbiocidal activities of this resin were demonstrated using single-species biofilms of Streptococcus mutans (ATCC 36558), Actinomyces naeslundii (ATCC 12104) and Candida albicans (ATCC 90028). Improved mechanical properties after hydration provided the proof-of-concept that QAMS-incorporated resin exhibits self-repair potential via water-induced condensation of organic modified silicate (ormosil) phases within the polymerized resin matrix.
PMCID: PMC3580770  PMID: 22659173
Quaternary ammonium; Organic modified silicate; Antimicrobial; Sol-gel technique; Self-repair
6.  An ORMOSIL-Containing Orthodontic Acrylic Resin with Concomitant Improvements in Antimicrobial and Fracture Toughness Properties 
PLoS ONE  2012;7(8):e42355.
Global increase in patients seeking orthodontic treatment creates a demand for the use of acrylic resins in removable appliances and retainers. Orthodontic removable appliance wearers have a higher risk of oral infections that are caused by the formation of bacterial and fungal biofilms on the appliance surface. Here, we present the synthetic route for an antibacterial and antifungal organically-modified silicate (ORMOSIL) that has multiple methacryloloxy functionalities attached to a siloxane backbone (quaternary ammonium methacryloxy silicate, or QAMS). By dissolving the water-insoluble, rubbery ORMOSIL in methyl methacrylate, QAMS may be copolymerized with polymethyl methacrylate, and covalently incorporated in the pressure-processed acrylic resin. The latter demonstrated a predominantly contact-killing effect on Streptococcus mutans ATCC 36558 and Actinomyces naselundii ATCC 12104 biofilms, while inhibiting adhesion of Candida albicans ATCC 90028 on the acrylic surface. Apart from its favorable antimicrobial activities, QAMS-containing acrylic resins exhibited decreased water wettability and improved toughness, without adversely affecting the flexural strength and modulus, water sorption and solubility, when compared with QAMS-free acrylic resin. The covalently bound, antimicrobial orthodontic acrylic resin with improved toughness represents advancement over other experimental antimicrobial acrylic resin formulations, in its potential to simultaneously prevent oral infections during appliance wear, and improve the fracture resistance of those appliances.
PMCID: PMC3411672  PMID: 22870322
7.  Mass Spectrometry Accelerates Membrane Protein Analysis 
Trends in biochemical sciences  2011;36(7):388-396.
Cellular membranes are composed of proteins and glyco- and phospholipids and play an indispensible role in maintaining cellular integrity and homeostasis by physically restricting biochemical processes within cells and providing protection. Membrane proteins perform many essential functions, which include operating as transporters, adhesion-anchors, receptors, and enzymes. Recent advancements in proteomic mass spectrometry have resulted in substantial progress towards the determination of the plasma membrane (PM) proteome, resolution of membrane protein topology, establishment of numerous receptor protein complexes, identification of ligand–receptor pairs, and the elucidation of signaling networks originating at the PM. Here we discuss the recent accelerated success of discovery-based proteomic pipelines for the establishment of a complete membrane proteome.
PMCID: PMC3222592  PMID: 21616670
8.  Interaction between FIP5 and SNX18 regulates epithelial lumen formation 
The Journal of Cell Biology  2011;195(1):71-86.
The Rab11 GTPase-binding protein FIP5 collaborates with the sorting nexin 18 to transport proteins to the apical surface and to tubulate membranes during epithelial apical lumen formation.
During the morphogenesis of the epithelial lumen, apical proteins are thought to be transported via endocytic compartments to the site of the forming lumen, although the machinery mediating this transport remains to be elucidated. Rab11 GTPase and its binding protein, FIP5, are important regulators of polarized endocytic transport. In this study, we identify sorting nexin 18 as a novel FIP5-interacting protein and characterize the role of FIP5 and SNX18 in epithelial lumen morphogenesis. We show that FIP5 mediates the transport of apical proteins from apical endosomes to the apical plasma membrane and, along with SNX18, is required for the early stages of apical lumen formation. Furthermore, both proteins bind lipids, and FIP5 promotes the capacity of SNX18 to tubulate membranes, which implies a role for FIP5 and SNX18 in endocytic carrier formation and/or scission. In summary, the present findings support the hypothesis that this FIP5-SNX18 complex plays a pivotal role in the polarized transport of apical proteins during apical lumen initiation in epithelial cells.
PMCID: PMC3187708  PMID: 21969467
9.  Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline 
Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.
PMCID: PMC3418851  PMID: 22454539
10.  Rab11-FIP3 is a cell cycle-regulated phosphoprotein 
BMC Cell Biology  2012;13:4.
Rab11 and its effector molecule, Rab11-FIP3 (FIP3), associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. FIP3 also controls recycling endosome distribution during interphase. Here, we examine whether phosphorylation of FIP3 is involved in these activities.
We identify four sites of phosphorylation of FIP3 in vivo, S-102, S-280, S-347 and S-450 and identify S-102 as a target for Cdk1-cyclin B in vitro. Of these, we show that S-102 is phosphorylated in metaphase and is dephosphorylated as cells enter telophase. Over-expression of FIP3-S102D increased the frequency of binucleate cells consistent with a role for this phospho-acceptor site in cytokinesis. Mutation of S-280, S-347 or S-450 or other previously identified phospho-acceptor sites (S-488, S-538, S-647 and S-648) was without effect on binucleate cell formation and did not modulate the distribution of FIP3 during the cell cycle. In an attempt to identify a functional role for FIP3 phosphorylation, we report that the change in FIP3 distribution from cytosolic to membrane-associated observed during progression from anaphase to telophase is accompanied by a concomitant dephosphorylation of FIP3. However, the phospho-acceptor sites identified here did not control this change in distribution.
Our data thus identify FIP3 as a cell cycle regulated phosphoprotein and suggest dephosphorylation of FIP3 accompanies its translocation from the cytosol to membranes during telophase. S102 is dephosphorylated during telophase; mutation of S102 exerts a modest effect on cytokinesis. Finally, we show that de/phosphorylation of the phospho-acceptor sites identified here (S-102, S-280, S-347 and S-450) is not required for the spatial control of recycling endosome distribution or function.
PMCID: PMC3310825  PMID: 22401586
Cytokinesis; Rab11-FIP3; Cdk1; Endosomes
11.  Quantitative improvements in peptide recovery at elevated chromatographic temperatures from μLC/MS analyses of brain using SRM mass spectrometry 
Analytical chemistry  2010;82(9):3435-3440.
Elevated chromatographic temperatures are well recognized to provide beneficial analytical effects. Previously, we demonstrated that elevated chromatographic temperature enhances the identification of hydrophobic peptides prepared from enriched membrane samples. Here, we quantitatively assess and compare the recovery of peptide analytes from both simple and complex tryptic peptide matrices using the SRM mass spectrometry. Our study demonstrates that elevated chromatographic temperature results in significant improvements in the magnitude of peptide recovery for both hydrophilic and hydrophobic peptides from both simple and complex peptide matrices. Importantly, the analytical benefits for quantitative measurements in whole mouse brain matrix are demonstrated, suggesting broad utility in the proteomic analyses of complex mammalian tissues. Any improvement in peptide recovery from chromatographic separations translates directly to the apparent sensitivity of downstream mass analysis in μLC-MS/MS based proteomic applications. Therefore, the incorporation of elevated chromatographic temperatures should result in significant improvements in peptide quantification as well as detection and identification.
PMCID: PMC2903057  PMID: 20373813
13.  FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network 
Molecular Biology of the Cell  2010;21(17):3041-3053.
This study shows that Rab11 and its binding protein FIP1 are required for retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN. We also demonstrate that Golgin-97 as a FIP1-binding protein and that this binding regulates the targeting of retrograde transport vesicles to the TGN.
Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.
PMCID: PMC2929997  PMID: 20610657
14.  HMG-CoA reductase inhibitors in kidney transplant recipients receiving tacrolimus: statins not associated with improved patient or graft survival 
BMC Nephrology  2010;11:5.
The beneficial effects of early statin use in kidney transplant recipients, especially those on tacrolimus-based immunosuppression, are not well established. We evaluated the predictors of statin use following kidney transplantation and examined its association with patient and allograft survival.
We examined 615 consecutive patients who underwent kidney transplant at our institution between January 1998 and January 2002. Statin use was assessed at baseline and 3, 6, 9, and 12 months following kidney transplant. Patients were followed for allograft and patient survival.
36% of the 615 kidney transplant recipients were treated with statin treatment. Statin use increased over the course of the study period. Older age, elevated body mass index, higher triglyceride levels, hypercholesterolemia, diabetes, history of myocardial infarction were associated with higher rates of statin use; elevated alkaline phosphatase levels and CMV IgG seropositivity were associated with less statin use. Older age, elevated BMI and hypercholesterolemia remained significant predictors of increased statin use after accounting for covariates using multiple regression. The early use of statins was not associated with improvements in unadjusted patient survival [HR 0.99; 95%CI 0.72-1.37] or graft survival [HR 0.97; 95% CI 0.76-1.24]. The risks of death and graft survival were not consistently reduced with exposure to statin using either adjusted models or propensity scores in Cox Proportional Hazards models.
In a kidney transplant population primarily receiving tacrolimus-based immunosuppression, early statin use was not associated with significantly improved graft or patient survival.
PMCID: PMC2855559  PMID: 20359353
15.  Quantitative strategies to fuel the merger of discovery and hypothesis-driven shotgun proteomics 
The ultimate goal of most shotgun proteomic pipelines is the discovery of novel biomarkers to direct the development of quantitative diagnostics for the detection and treatment of disease. Differential comparisons of biological samples identify candidate peptides that can serve as proxys of candidate proteins. While these discovery approaches are robust and fairly comprehensive, they have relatively low throughput. When merged with targeted mass spectrometry, this pipeline can fuel hypothesis-driven studies and the development of novel diagnostics and therapeutics.
PMCID: PMC3105289  PMID: 19398505
quantitative shotgun proteomics; biomarker discovery; targeted mass spectrometry; human tissue
16.  In Vitro Remineralization Effects of Grape Seed Extract on Artificial Root Caries 
Journal of dentistry  2008;36(11):900-906.
Grape seed extract (GSE) contains Proanthocyanidin (PA), which has been reported to strengthen collagen-based tissues by increasing collagen cross-links. We used an in vitro pH-cycling model to evaluate the effect of GSE on the remineralization of artificial root caries. Sound human teeth fragments obtained from the cervical portion of the root were stored in a demineralization solution for 96 hr at 37°C to induce artificial root caries lesions. The fragments were then divided into three treatment groups including: 6.5% GSE, 1,000 ppm fluoride (NaF), and a control (no treatment). The demineralized samples were pH-cycled through treatment solutions, acidic buffer and neutral buffer for 8 days at 6 cycles per day. The samples were subsequently evaluated using a microhardness tester; polarized light microscopy (PLM) and confocal laser scanning microscopy (CLSM). Data were analyzed using ANOVA and Fisher’s tests (p<0.05). GSE and fluoride significantly increased the microhardness of the lesions (p<0.05) when compared to a control group. PLM data revealed a significantly thicker mineral precipitation band on the surface layer of the GSE treated lesions when compared to the other groups (p>0.05), which was confirmed by CLSM. We concluded that grape seed extract positively affects the demineralization and/or remineralization processes of artificial root caries lesions, most likely through a different mechanism than that of Fluoride. Grape seed extract may be a promising natural agent for non-invasive root caries therapy.
PMCID: PMC2583354  PMID: 18819742
17.  A Shotgun Proteomic Method for the Identification of Membrane-Embedded Proteins and Peptides 
Journal of proteome research  2008;7(7):3028-3034.
Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome.
PMCID: PMC2765231  PMID: 18537282
18.  MudPIT Analysis: Application to Human Heart Tissue 
Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as the standard proteomic approach for separating proteins in a complex mixture, this technique has many drawbacks. These include a limited molecular mass range, poor separation of highly acidic or basic proteins, and exclusion of the majority of membrane proteins from analysis. Considering the important functions of many membrane proteins, such as receptors, ion transporters, signal transducers, and cell adhesion proteins, it is increasingly important that these proteins are not excluded during the global proteomic analysis of cellular systems. Multidimensional Protein Identification Technology (MudPIT) offers a gel-free alternative to 2D-PAGE for the analysis of both membrane and soluble proteins.
The goal of this chapter is to provide detailed methods for using MudPIT to profile both membrane and soluble proteins in complex unfractionated samples. Methods discussed will include tissue homogenization, sample preparation, MudPIT, data analysis, and an application for the analysis of unfractionated total tissue homogenate from human heart.
PMCID: PMC2765120  PMID: 19153700
MudPIT; 2D-PAGE; proteomics; membrane proteins; human heart explants
19.  High Quality Catalog of Proteotypic Peptides from Human Heart 
Journal of proteome research  2008;7(11):5055-5061.
Proteomics research is beginning to expand beyond the more traditional shotgun analysis of protein mixtures to include targeted analyses of specific proteins using mass spectrometry. Integral to the development of a robust assay based on targeted mass spectrometry is prior knowledge of which peptides provide an accurate and sensitive proxy of the originating gene product (i.e., proteotypic peptides). To develop a catalog of “proteotypic peptides” in human heart, TRIzol extracts of left-ventricular tissue from nonfailing and failing human heart explants were optimized for shotgun proteomic analysis using Multidimensional Protein Identification Technology (MudPIT). Ten replicate MudPIT analyses were performed on each tissue sample and resulted in the identification of 30 605 unique peptides with a q-value ≤ 0.01, corresponding to 7138 unique human heart proteins. Experimental observation frequencies were assessed and used to select over 4476 proteotypic peptides for 2558 heart proteins. This human cardiac data set can serve as a public reference to guide the selection of proteotypic peptides for future targeted mass spectrometry experiments monitoring potential protein biomarkers of human heart diseases.
PMCID: PMC2765113  PMID: 18803417
proteotypic peptides; targeted mass spectrometry; human heart explant; dilated cardiomyopathy; MudPIT
20.  Chromatographic benefits of elevated temperature for the proteomic analysis of membrane proteins 
Proteomics  2008;8(19):3956-3964.
Integral membrane proteins (IMPs) perform crucial cellular functions and are the primary targets for most pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains and their intimate association with lipids makes them difficult to handle. Multiple proteomics platforms that include LC separations have been reported for the high-throughput profiling of complex protein samples. However, there are still many challenges to overcome for proteomic analyses of IMPs, especially as compared to their soluble counterparts. In particular, considerations for the technical challenges associated with chromatographic separations are just beginning to be investigated. Here, we review the benefits of using elevated temperatures during LC for the proteomic analysis of complex membrane protein samples.
PMCID: PMC2765112  PMID: 18780350
Liquid chromatography; Microcapillary; Shotgun; Temperature
21.  DNA Microarray and Proteomic Strategies for Understanding Alcohol Action 
This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California. The organizer was James M. Sikela, and he and Michael F. Miles were chairs. The presentations were (1) Genomewide Surveys of Gene Copy Number Variation in Human and Mouse: Implications for the Genetics of Alcohol Action, by James M. Sikela; (2) Regional Differences in the Regulation of Brain Gene Expression: Relevance to the Detection of Genes Associated with Alcohol-Related Traits, by Robert Hitzemann; (3) Identification of Ethanol Quantitative Trait Loci Candidate Genes by Expression Profiling in Inbred Long Sleep/Inbred Short Sleep Congenic Mice, by Robnet T. Kerns; and (4) Quantitative Proteomic Analysis of AC7-Modified Mice, by Kathleen J. Grant.
PMCID: PMC2709534  PMID: 16573589
Array-Based Comparative Genomic Hybridization; Gene Copy Number; Microarrays; Gene Expression Profiling; Alcohol-Related QTL; Proteomics; Adenylyl Cyclase
22.  15N Metabolic Labeling of Mammalian Tissue with Slow Protein Turnover 
Journal of proteome research  2007;6(5):2005-2010.
We previously reported the metabolic 15N labeling of a rat where enrichment ranged from 94% to 74%. We report here an improved labeling strategy which generates 94% 15N enrichment throughout all tissues of the rat. A high 15N enrichment of the internal standard is necessary for accurate quantitation, and thus, this approach will allow quantitative mass spectrometry analysis of animal models of disease targeting any tissue.
PMCID: PMC2527585  PMID: 17375949
23.  The Role of GRASP55 in Golgi Fragmentation and Entry of Cells into Mitosis 
Molecular Biology of the Cell  2008;19(6):2579-2587.
GRASP55 is a Golgi-associated protein, but its function at the Golgi remains unclear. Addition of full-length GRASP55, GRASP55-specific peptides, or an anti-GRASP55 antibody inhibited Golgi fragmentation by mitotic extracts in vitro, and entry of cells into mitosis. Phospho-peptide mapping of full-length GRASP55 revealed that threonine 225 and 249 were mitotically phosphorylated. Wild-type peptides containing T225 and T249 inhibited Golgi fragmentation and entry of cells into mitosis. Mutant peptides containing T225E and T249E, in contrast, did not affect Golgi fragmentation and entry into mitosis. These findings reveal a role of GRASP55 in events leading to Golgi fragmentation and the subsequent entry of cell into mitosis. Surprisingly, however, under our experimental conditions, >85% knockdown of GRASP55 did not affect the overall organization of Golgi organization in terms of cisternal stacking and lateral connections between stacks. Based on our findings we suggest that phosphorylation of GRASP55 at T225/T249 releases a bound component, which is phosphorylated and necessary for Golgi fragmentation. Thus, GRASP55 has no role in the organization of Golgi membranes per se, but it controls their fragmentation by regulating the release of a partner, which requires a G2-specific phosphorylation at T225/T249.
PMCID: PMC2397314  PMID: 18385516
24.  Proteomic Solutions for Analytical Challenges Associated With Alcohol Research 
Alcohol Research & Health  2008;31(3):251-255.
PMCID: PMC3860482  PMID: 23584870
Alcohol dependence; genetic theory of alcohol and other drug use; genetic trait; brain; animal models; proteins; protein analysis; proteomics; mass spectrometry; peptides
25.  Chlamydial GroEL Autoregulates Its Own Expression through Direct Interactions with the HrcA Repressor Protein 
Journal of Bacteriology  2005;187(21):7535-7542.
In the pathogenic bacterium Chlamydia trachomatis, a transcriptional repressor, HrcA, regulates the major heat shock operons, dnaK and groE. Cellular stress causes a transient increase in transcription of these heat shock operons through relief of HrcA-mediated repression, but the pathway leading to derepression is unclear. Elevated temperature alone is not sufficient, and it is hypothesized that additional chlamydial factors play a role. We used DNA affinity chromatography to purify proteins that interact with HrcA in vivo and identified a higher-order complex consisting of HrcA, GroEL, and GroES. This endogenous HrcA complex migrated differently than recombinant HrcA, but the complex could be disrupted, releasing native HrcA that resembled recombinant HrcA. In in vitro assays, GroEL increased the ability of HrcA to bind to the CIRCE operator and to repress transcription. Other chlamydial heat shock proteins, including the two additional GroEL paralogs present in all chlamydial species, did not modulate HrcA activity.
PMCID: PMC1272993  PMID: 16237037

Results 1-25 (29)