Proteomic Techniques in the Physiological Proteomics Core Facility

poster abstractA new software package, IdentiQuantXL, has been developed in the Physiological Proteomics Core Facility to provide large-scale protein identification and label-free quantification using either low or high resolution LC-MS/MS data. Though many software packages have been developed to perform label-free quantification of proteins in complex biological samples using peptide intensities generated by liquid chromatography - tandem mass spectrometry (LC-MS/MS), two important issues hinder the use of peptide intensity measurements: (i) It is difficult to accurately determine the retention time of each peptide peak, especially for low resolution data, and (ii) many peptides cannot be used for protein quantification. To address these two key issues, we have developed a new method to enable accurate peptide peak retention time determination and multiple filters to eliminate unqualified peptides for protein quantification. Repeatability and linearity have been tested using ion trap-derived low resolution data from six very different samples, i.e., standard peptides, kidney tissue lysates, HT29-MTX cell lysates, depleted human serum, human serum albumin-bound proteins, and standard proteins spiked in kidney tissue lysates. In all these unique experiments, at least 90.8% of proteins (up to 1,390) had CVs ≤ 3 0% across 10 technique replicates, and at least 92.1% of proteins (up to 2,013) had R2 ≥ 0.9500 across 7 concentrations. The performance of our strategy was verified using identical amounts of standard protein (lysozyme) spiked in complex biological samples (cell culture media containing secreted proteins) with a CV of 8.6% across eight injections. The excellent performance was further confirmed by comparing label-free mass spectrometry to Western blot detection of prolactin, which was decreased 17.1fold in dwarfed mice compared to wild-type using the label-free quantification strategy and very low or undetectable using Western blot. The results indicate that our new platform, named IdentiQuantXL, accurately quantifies thousands of peptides and proteins in complex samples. It has been applied in the aqueous humor proteome in patients with Fuchs endothelial corneal dystrophy. While many software packages focus only on high resolution data, our strategy is designed for both high and low resolution data. Consequently, it is very useful for data generated by low resolution mass spectrometers such as the LTQ, especially when the dynamic exclusion of ions in data acquisition is enabled to obtain more MS/MS fragments of low-abundance peptides to maximally identify proteins in a complex biological sample. Supported by NIEHS RC2ES018810 and NIGMS R01GM08521

Mathematical Nanotoxicoproteomics: Quantitative Characterization of Effects of Multi-walled Carbon Nanotubes (MWCNT) and TiO2 Nanobelts (TiO2-NB) on Protein Expression Patterns in Human Intestinal Cells

Background: Various applications of nanosubstances in industrial and consumer goods sectors are growing rapidly because of their useful chemical and physical properties. Objectives: Assessment of hazard posed by exposure to nanosubstances is essential for the protection of human and ecological health. Methods: We analyzed the proteomics patterns of Caco-2/HT29-MTX cells in co-culture exposed for three and twenty four hours to two kinds of nanoparticles: multi-walled carbon nanotubes (MWCNT) and TiO2 nanobelts (TiO2-NB). For each nanosubstance cells were exposed to two concentrations of the material before carrying out proteomics analyses: 10 μg and 100 μg. In each case over 3000 proteins were identified. A mathematically based similarity index, which measures the changes in abundances of cellular proteins that are highly affected by exposure to the nanosubstances, was used to characterize toxic effects of the nanomaterials. Results: We identified 8 and 25 proteins, which are most highly affected by MWCNT and TiO2-NB, respectively. These proteins may be responsible for specific response of cells to the nanoparticles. Further 14 reported proteins are affected by either of the two nanoparticles and they are probably related to nonspecific toxic response of the cells. Conclusion: The similarity methods proposed in this paper may be useful in the management and visualization of the large amount of data generated by proteomics technologies

Delineation of Molecular Pathways Involved in Cardiomyopathies Caused by Troponin T Mutations

Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca(2+) sensitivity of the myofilament; however, the R278C mutation does not alter Ca(2+) sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism

Functional Effects of Nanoparticle Exposure on Calu-3 Airway Epithelial Cells

High concentrations of manufactured carbon nanoparticles (CNP) are known to cause oxidative stress, inflammatory responses and granuloma formation in respiratory epithelia. To examine the effects of lower, more physiologically relevant concentrations, the human airway epithelial cell line, Calu-3, was used to evaluate potential alterations in transepithelial permeability and cellular function of airway epithelia after exposure to environmentally realistic concentrations of carbon nanoparticles. Three common carbon nanoparticles, fullerenes, single- and multi-wall carbon nanotubes (SWCNT, MWCNT) were used in these experiments. Electrophysiological measurements were performed to assay transepithelial electrical resistance (TEER) and epinephrine-stimulated chloride (Cl(-)) ion secretion of epithelial cell monolayers that had been exposed to nanoparticles for three different times (1 h, 24 h and 48 h) and over a 7 log unit range of concentrations. Fullerenes did not have any effect on the TEER or stimulated ion transport. However, the carbon nanotubes (CNT) significantly decreased TEER and inhibited epinephrine-stimulated Cl(-) secretion. The changes were time dependent and at more chronic exposures caused functional effects which were evident at concentrations substantially lower than have been previously examined. The functional changes manifested in response to physiologically relevant exposures would inhibit mucociliary clearance mechanisms and compromise the barrier function of airway epithelia

Distinctive and pervasive alterations in aqueous humor protein composition following different types of glaucoma surgery

PURPOSE: To investigate whether specific glaucoma surgeries are associated with differences in aqueous humor protein concentrations compared to eyes without filters. METHODS: In this cross-sectional study, aqueous humor samples were prospectively collected from control subjects who underwent routine cataract surgery (n=14) and from patients who had different glaucoma filters: Baerveldt aqueous shunt (n=6), Ahmed aqueous shunt (n=6), trabeculectomy (n=5), and Ex-Press trabeculectomy (n=3). Total protein concentrations were determined with Bradford assay. Tryptic digests were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins were identified with high confidence using stringent criteria and were quantitatively compared with a label-free platform. Relative protein quantities were compared across groups with ANOVA. Post hoc pair-wise comparisons were adjusted for multiple comparisons. RESULTS: Compared to the control eyes, the aqueous humor protein concentration was increased approximately tenfold in the Ahmed and Baerveldt eyes and fivefold in the trabeculectomy and Ex-Press eyes. Overall, 718 unique proteins, splice variants, or isoforms were identified. No differences in the protein concentrations were detected between the Baerveldt and Ahmed groups. Likewise, the trabeculectomy and Ex-Press groups were remarkably similar. Therefore, the aqueous shunt groups were pooled, and the trabeculectomy groups were pooled for a three-way comparison with the controls. More than 500 proteins differed significantly in relative abundance (ANOVA p<0.01) among the control, aqueous shunt, and trabeculectomy groups. Functional analyses suggested these alterations in relative protein abundance affected dozens of signaling pathways. CONCLUSIONS: Different glaucoma surgical procedures were associated with marked increases in the aqueous humor protein concentration and distinctive changes in the relative abundance of numerous proteins involved in multiple signaling pathways

Cancer and Chemotherapy Contribute to Muscle Loss by Activating Common Signaling Pathways

Cachexia represents one of the primary complications of colorectal cancer due to its effects on depletion of muscle and fat. Evidence suggests that chemotherapeutic regimens, such as Folfiri, contribute to cachexia-related symptoms. The purpose of the present study was to investigate the cachexia signature in different conditions associated with severe muscle wasting, namely Colon-26 (C26) and Folfiri-associated cachexia. Using a quantitative LC-MS/MS approach, we identified significant changes in 386 proteins in the quadriceps muscle of Folfiri-treated mice, and 269 proteins differentially expressed in the C26 hosts (p < 0.05; -1.5 ≥ fold change ≥ +1.5). Comparative analysis isolated 240 proteins that were modulated in common, with a large majority (218) that were down-regulated in both experimental settings. Interestingly, metabolic (47.08%) and structural (21.25%) proteins were the most represented. Pathway analysis revealed mitochondrial dysfunctions in both experimental conditions, also consistent with reduced expression of mediators of mitochondrial fusion (OPA-1, mitofusin-2), fission (DRP-1) and biogenesis (Cytochrome C, PGC-1α). Alterations of oxidative phosphorylation within the TCA cycle, fatty acid metabolism, and Ca(2+) signaling were also detected. Overall, the proteomic signature in the presence of both chemotherapy and cancer suggests the activation of mechanisms associated with movement disorders, necrosis, muscle cell death, muscle weakness and muscle damage. Conversely, this is consistent with the inhibition of pathways that regulate nucleotide and fatty acid metabolism, synthesis of ATP, muscle and heart function, as well as ROS scavenging. Interestingly, strong up-regulation of pro-inflammatory acute-phase proteins and a more coordinated modulation of mitochondrial and lipidic metabolisms were observed in the muscle of the C26 hosts that were different from the Folfiri-treated animals. In conclusion, our results suggest that both cancer and chemotherapy contribute to muscle loss by activating common signaling pathways. These data support the undertaking of combination strategies that aim to both counteract tumor growth and reduce chemotherapy side effects

Comparative Proteomics and Biological Effects of Functionalized Carbon Nanotubes in Intestinal Cell Co-culture

poster abstract“Carbon nanotubes (CNTs) possess unique electrical, mechanical, and thermal properties, with potential applications in the electronics, catalysts, polymer composites, aerospace, and other industries. CNTs are also being developed for a broad range of applications in biomedicine, including oral drug delivery. Functionalized, water dispersible CNTs (fCNTS) can be expected to enter the digestive tract and exert biological effects on its barrier epithelial cells. To characterize these effects, we developed an in vitro model of the large intestinal tract using a coculture of Caco-2 (75%) and HT29-MTX (25%, mucus secreting) cells, and exposed these cells to functionalized single-walled (SWNT) and multi-walled (MWNT) carbon nanotubes at realistic concentrations (500 pg/mL and 10 µg/mL; 48 h). Protein expression was analyzed using our recently developed label-free quantitative mass spectrometry (LFQMS) platform, IdentiQuantXL™, while typical toxicological endpoint assays were used to characterize various cellular responses. LFQMS identified 5,007 unique protein database entries, from which 4,200 proteins were considered qualified for quantitation. These proteins represented 1,978 protein groups (containing isoforms, splice-variants, etc). Differences in expression were calculated by ANOVA (P<0.001) and post hoc Holm Sidak comparisons (P<0.05). fCNT significantly altered protein expression in a moderate number of proteins, the extent and type of which were fCNT specific. Only 13 proteins were universally altered by all exposures (except 500 pg/mL COOHSWNT which had no effect), and these represent a broad range of cellular functions. Bioinformatic analysis using the Gene Ontology Database and Ingenuity Pathway Analysis revealed statistically significant protein associations with a broad range of functional networks and signaling/metabolic pathways. Again, little overlap between fCNT was observed. None of the exposures was associated with overt toxicity or proinflammatory response. The results suggest that significant biological effects result from fCNT exposure, responses that are specific to CNT-type and dose, but occurring in the absence of toxicity or irritation. Supported by NIEHS RC2ES018810.

Structural and Functional Analyses of Liver Cysts from the BALB/c-cpk Mouse Model of Polycystic Kidney Disease

Liver cysts arising from hepatic bile ducts are a common extra-renal pathology associated with both autosomal dominant and recessive polycystic kidney disease in humans. To elucidate the functional and structural changes inherent in cyst formation and growth, hepatic bile duct epithelia were isolated from the BALB/ c-cpk mouse model of polycystic kidney disease. Light and transmission electron microscopy revealed substantial fibrosis in the basal lamina surrounding hepatic bile duct cysts isolated from heterozygous (BALB/c-cpk/+) and homozygous (BALB/c-cpk/cpk) animals. Scanning electron microscopy and length analysis of normal, precystic and cystic bile ducts provided the unique observation that primary cilia in cholangiocytes isolated from bile ducts and cysts of animals expressing the mutated cpk gene had lengths outside the minimal and maximal ranges of those in cells lining bile ducts of wild-type animals. Based on the hypothesis that PKD is one of several diseases characterized as ciliopathies, this abnormal variability in the length of the primary cilia may have functional implications. Electrophysiological analyses of freshly isolated cysts indicate that the amiloride-sensitive epithelial Na(+) channel (ENaC) is inactive/absent and cAMP-mediated anion secretion is the electrogenic transport process contributing to cyst fluid accumulation. Anion secretion can be stimulated by the luminal stimulation of adenylyl cyclase

Proteomic profiling of halloysite clay nanotube exposure in intestinal cell co-culture

Halloysite is aluminosilicate clay with a hollow tubular structure with nanoscale internal and external diameters. Assessment of halloysite biocompatibility has gained importance in view of its potential application in oral drug delivery. To investigate the effect of halloysite nanotubes on an in vitro model of the large intestine, Caco-2/HT29-MTX cells in monolayer co-culture were exposed to nanotubes for toxicity tests and proteomic analysis. Results indicate that halloysite exhibits a high degree of biocompatibility characterized by an absence of cytotoxicity, in spite of elevated pro-inflammatory cytokine release. Exposure-specific changes in expression were observed among 4081 proteins analyzed. Bioinformatic analysis of differentially expressed protein profiles suggest that halloysite stimulates processes related to cell growth and proliferation, subtle responses to cell infection, irritation and injury, enhanced antioxidant capability, and an overall adaptive response to exposure. These potentially relevant functional effects warrant further investigation in in vivo models and suggest that chronic or bolus occupational exposure to halloysite nanotubes may have unintended outcomes

Increasing serum pre-adipocyte factor-1 (Pref-1) correlates with decreased body fat, increased free fatty acids, and level of recent alcohol consumption in excessive alcohol drinkers

Patients with alcoholic liver disease have been reported to have a significantly lower percentage of body fat (%BF) than controls. The mechanism for the reduction in %BF in heavy alcohol users has not been elucidated. In adipose tissue, Pref-1 is specifically expressed in pre-adipocytes but not in adipocytes. Pref-1 inhibits adipogenesis and elevated levels are associated with reduced adipose tissue mass. We investigated the association between serum Pref-1 and %BF, alcohol consumption, and serum free fatty acids (FFA) in a well-characterized cohort of heavy alcohol users compared to controls. One hundred forty-eight subjects were prospectively recruited. The Time Line Follow-Back (TLFB) questionnaire was used to quantify the amount of alcohol consumed over the 30-day period before their enrollment. Anthropometric measurements were performed to calculate %BF. Serum Pref-1 and FFA were measured. Fifty-one subjects (mean age 32 ± 9 years, 88% men) were non-excessive drinkers whereas 97 were excessive drinkers (mean age 41 ± 18 years, 69% men). Compared to non-excessive drinkers, individuals with excessive drinking had significantly higher levels of Pref-1 (p<0.01), FFA (p < 0.001), and lower %BF (p = 0.03). Serum levels of Pref-1 were associated with the amount of alcohol consumed during the previous 30 days. Serum Pref-1 was negatively correlated with %BF, but positively associated with serum FFA. Our data suggest that elevated Pref-1 levels in excessive drinkers might inhibit the expansion of adipose tissue, decreasing %BF in alcoholics. Further work is needed to validate these findings and to better understand the role of Pref-1 and its clinical significance in subjects with heavy alcohol use