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1.  Proteomic characterization of serine hydrolase activity and composition in normal urine 
Clinical Proteomics  2013;10(1):17.
Background
Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis.
Methods
Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine.
Results
Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state.
Conclusions
This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.
doi:10.1186/1559-0275-10-17
PMCID: PMC4225696  PMID: 24237849
Activity-based protein profiling; Catabolomics; Fluorophosphonate probe; Mass spectrometry
2.  Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression 
BMC Microbiology  2012;12:214.
Background
Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.
Results
Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.
Conclusions
Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.
doi:10.1186/1471-2180-12-214
PMCID: PMC3492117  PMID: 22994686
3.  Requirement of Podocalyxin in TGF-Beta Induced Epithelial Mesenchymal Transition 
PLoS ONE  2011;6(4):e18715.
Epithelial mesenchymal transition (EMT) is characterized by the development of mesenchymal properties such as a fibroblast-like morphology with altered cytoskeletal organization and enhanced migratory potential. We report that the expression of podocalyxin (PODXL), a member of the CD34 family, is markedly increased during TGF-β induced EMT. PODXL is enriched on the leading edges of migrating A549 cells. Silencing of podocalyxin expression reduced cell ruffle formation, spreading, migration and affected the expression patterns of several proteins that normally change during EMT (e.g., vimentin, E-cadherin). Cytoskeletion assembly in EMT was also found to be dependent on the production of podocalyin. Compositional analysis of podocalyxin containing immunoprecipitates revealed that collagen type 1 was consistently associated with these isolates. Collagen type 1 was also found to co-localize with podocalyxin on the leading edges of migrating cells. The interactions with collagen may be a critical aspect of podocalyxin function. Podocalyxin is an important regulator of the EMT like process as it regulates the loss of epithelial features and the acquisition of a motile phenotype.
doi:10.1371/journal.pone.0018715
PMCID: PMC3075272  PMID: 21533279
4.  Response of Primary Human Airway Epithelial Cells to Influenza Infection: A Quantitative Proteomic Study 
Journal of Proteome Research  2012;11(8):4132-4146.
Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify host proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC–MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells.
doi:10.1021/pr300239r
PMCID: PMC3411195  PMID: 22694362
Influenza virus; Primary cells; Quantitative proteomics; Host cell response; SILAC

Results 1-4 (4)