Biomedical subjects

Rong-Fong Shen

Publications and source records attributed to Rong-Fong Shen.

18 recordsLinked to original sources

PubMed2006

Proteomic identification of proteins associated with the osmoregulatory transcription factor TonEBP/OREBP: functional effects of Hsp90 and PARP-1.

Hypertonicity (e.g., high NaCl) activates the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP), increasing transcription of protective genes. In the present studies, by stably expressing amino acids 1-547 of TonEBP/OREBP in HEK 293 cells and immunoprecipitating it plus associated proteins from the nuclei of cells exposed to high NaCl, we identify 14 proteins that are physically associated with TonEBP/OREBP. The associated proteins fall into several classes: 1) DNA-dependent protein kinase, both its catalytic subunit and regulatory subunit, Ku86; 2) RNA helicases, namely RNA helicase A, nucleolar RNA helicase II/Gu, and DEAD-box RNA helicase p72; 3) small or heterogeneous nuclear ribonucleoproteins (snRNPs or hnRNPs), namely U5 snRNP-specific 116 kDa protein, U5 snRNP-specific 200 kDa protein, hnRNP U, hnRNP M, hnRNP K, and hnRNP F; 4) heat shock proteins, namely Hsp90beta and Hsc70; and 5) poly(ADP-ribose) polymerase-1 (PARP-1). We confirm identification of most of the proteins by Western analysis and also demonstrate by electrophoretic mobility-shift assay that they are present in the large complex that binds specifically along with TonEBP/OREBP to its cognate DNA element. In addition, we find that PARP-1 and Hsp90 modulate TonEBP/OREBP activity. PARP-1 expression reduces TonEBP/OREBP transcriptional activity and the activity of its transactivating domain. Hsp90 enhances those activities and sustains the increased abundance of TonEBP/OREBP protein in cells exposed to high NaCl.

Aldehyde Reductase↗

PubMed2006

Expression of SUMO-2/3 induced senescence through p53- and pRB-mediated pathways.

Three highly homologous small ubiquitin-related modifier (SUMO) proteins have been identified in mammals. Modifications of proteins by SUMO-1 have been shown to regulate transcription, nucleocytoplasmic transport, protein stability, and protein-protein interactions. Relative to SUMO-1, little is known about the functions of SUMO-2 or SUMO-3 (referred to as SUMO-2/3). Here, stable cell lines overexpressing processed forms of SUMO-2/3 (SUMO-2/3GG) as well as their non-conjugatable derivatives, SUMO-2/3DeltaGG, were established. Cells overexpressing SUMO-2/3GG showed a premature senescence phenotype as revealed by cellular morphology and senescence-associated galactosidase activity. The senescence pathway protein p21 was up-regulated in cells overexpressing SUMO-2/3GG. In contrast, cells overexpressing non-conjugatable forms of SUMO-2/3DeltaGG showed neither an apparent senescent phenotype nor elevated p21. Both p53 and pRB were found to be modified by SUMO-2/3. Site-directed mutagenesis studies showed that Lys-386 of p53, the SUMO-1 modification site, is also the modification site for SUMO-2/3. In addition, H2O2 treatment of untransfected cells caused an increase in p53 sumoylation by SUMO-2/3, whereas that by SUMO-1 remained unchanged. Moreover, knocking down tumor suppressor proteins p53 or pRB using small interfering RNA significantly alleviated the premature senescence phenotypes in SUMO-2/3GG overexpressing cells. Together, our results reveal that p53 and pRB can be sumoylated by SUMO-2/3 in vivo, and such modification of p53 and pRB may play roles in premature senescence and stress response.

Animals↗

PubMed2006

Automated quantification tool for high-throughput proteomics using stable isotope labeling and LC-MSn.

LC-MSn has become a popular option for high-throughput quantitative proteomics, thanks to the availability of stable-isotope labeling reagents. However, the vast quantity of data generated from LC-MSn continues to make the postacquisition quantification analyses challenging, especially in experiments involving multiple samples per experimental condition. To facilitate data analysis, we developed a computer program, QUIL, for automated protein quantification. QUIL accounts for the dynamic nature of spectral background and subtracts this background accordingly during ion chromatogram reconstruction. For elution profile identification, QUIL minimizes the inclusion of coeluted neighbor peaks, yet tolerates imperfect peak shapes. Outlier-resistant methods have been implemented for better protein ratio estimation. The utility of QUIL was validated by quantitative analyses of a standard protein as well as complex protein mixtures, which were labeled with cICAT or 18O and analyzed using LCQ, LTQ, or FT-ICR instruments. For samples that no prior knowledge of relative protein quantities was available, Western blotting was performed for confirmation. For the standard protein, the coefficient of variation (CV) of peptide ratio estimation was 6%. For complex mixtures, the median CV for protein ratio calculations was less than 10%. Computed protein abundance ratios exhibited a relatively high degree of correlation with those obtained from Western blot analyses. Compared with a widely used commercial software tool, QUIL showed improvement in ion chromatogram construction and peak integration and significantly reduced relative errors in abundance ratio assessment.

Animals↗

PubMed2006

LC-MS/MS analysis of apical and basolateral plasma membranes of rat renal collecting duct cells.

We used biotinylation and streptavidin affinity chromatography to label and enrich proteins from apical and basolateral membranes of rat kidney inner medullary collecting ducts (IMCDs) prior to LC-MS/MS protein identification. To enrich apical membrane proteins and bound peripheral membrane proteins, IMCDs were perfusion-labeled with primary amine-reactive biotinylation reagents at 2 degrees C using a double barreled pipette. The perfusion-biotinylated proteins and proteins bound to them were isolated with CaptAvidin-agarose beads, separated with SDS-PAGE, and sliced into continuous gel pieces for LC-MS/MS protein identification (LTQ, Thermo Electron Corp.). 17 integral and glycosylphosphatidylinositol (GPI)-linked membrane proteins and 44 non-integral membrane proteins were identified. Immunofluorescence confocal microscopy confirmed ACVRL1, H(+)/K(+)-ATPase alpha1, NHE2, and TauT expression in the IMCDs. Basement membrane and basolateral membrane proteins were biotinylated via incubation of IMCD suspensions with biotinylation reagents on ice. 23 integral and GPI-linked membrane proteins and 134 non-integral membrane proteins were identified. Analyses of non-integral membrane proteins preferentially identified in the perfusion-biotinylated and not in the incubation-biotinylated IMCDs revealed protein kinases, scaffold proteins, SNARE proteins, motor proteins, small GTP-binding proteins, and related proteins that may be involved in vasopressin-stimulated AQP2, UT-A1, and ENaC regulation. A World Wide Web-accessible database was constructed of 222 membrane proteins (integral and GPI-linked) from this study and prior studies.

Animals↗

PubMed2006

The mammalian target of rapamycin (mTOR) pathway regulates mitochondrial oxygen consumption and oxidative capacity.

Metabolic rate and the subsequent production of reactive oxygen species are thought to contribute to the rate of aging in a wide range of species. The target of rapamycin (TOR) is a well conserved serine/threonine kinase that regulates cell growth in response to nutrient status. Here we demonstrate that in mammalian cells the mammalian TOR (mTOR) pathway plays a significant role in determining both resting oxygen consumption and oxidative capacity. In particular, we demonstrate that the level of complex formation between mTOR and one of its known protein partners, raptor, correlated with overall mitochondrial activity. Disruption of this complex following treatment with the mTOR pharmacological inhibitor rapamycin lowered mitochondrial membrane potential, oxygen consumption, and ATP synthetic capacity. Subcellular fractionation revealed that mTOR as well as mTOR-raptor complexes can be purified in the mitochondrial fraction. Using two-dimensional difference gel electrophoresis, we further demonstrated that inhibiting mTOR with rapamycin resulted in a dramatic alteration in the mitochondrial phosphoproteome. RNA interference-mediated knockdown of TSC2, p70 S6 kinase (S6K1), raptor, or rictor demonstrates that mTOR regulates mitochondrial activity independently of its previously identified cellular targets. Finally we demonstrate that mTOR activity may play an important role in determining the relative balance between mitochondrial and non-mitochondrial sources of ATP generation. These results may provide insight into recent observations linking the TOR pathway to life span regulation of lower organisms.

Adenosine Triphosphate↗

PubMed2006

Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin-2 phosphorylation at two sites.

Protein phosphorylation plays a key role in vasopressin signaling in the renal-collecting duct. Large-scale identification and quantification of phosphorylation events triggered by vasopressin is desirable to gain a comprehensive systems-level understanding of this process. We carried out phosphoproteomic analysis of rat inner medullary collecting duct cells by using a combination of phosphopeptide enrichment by immobilized metal affinity chromatography and phosphorylation site identification by liquid chromatography-mass spectrometry(n) neutral loss scanning. A total of 714 phosphorylation sites on 223 unique phosphoproteins were identified from inner medullary collecting duct samples treated short-term with either calyculin A or vasopressin. A number of proteins involved in cytoskeletal reorganization, vesicle trafficking, and transcriptional regulation were identified. Previously unidentified phosphorylation sites were found for membrane proteins essential to collecting duct physiology, including eight sites among aquaporin-2 (AQP2), aquaporin-4, and urea transporter isoforms A1 and A3. Through label-free quantification of phosphopeptides, we identified a number of proteins that significantly changed phosphorylation state in response to short-term vasopressin treatment: AQP2, Bclaf1, LRRC47, Rgl3, and SAFB2. In the presence of vasopressin, AQP2 monophosphorylated at S256 and diphosphorylated AQP2 (pS256/261) increased in abundance, whereas AQP2 monophosphorylated at S261 decreased, raising the possibility that both sites are involved in vasopressin-dependent AQP2 trafficking. This study reveals the practicality of liquid chromatography-mass spectrometry(n) neutral loss scanning for large-scale identification and quantification of protein phosphorylation in the analysis of cell signaling in a native mammalian system.

Amino Acid Sequence↗

PubMed2006

A general approach for investigating enzymatic pathways and substrates for ubiquitin-like modifiers.

Ubiquitin-like modifiers (UBLs) contain ubiquitin homology domains and can covalently modify target proteins in a manner similar to ubiquitylation. In this study, we revealed a general proteomic approach to elucidate the enzymatic pathways and identify target proteins for three UBLs: SUMO-2, SUMO-3, and NEDD8. Expression plasmids containing the cDNAs of Myc/6xHis doubly-tagged processed or non-conjugatable forms of these UBLs were constructed. The constructed vectors were then used to transfect HEK 293 Tet-On cells, and stable cell lines expressing these UBLs and their mutants were established. The epitope-tagged proteins were purified by immunoprecipitation under native conditions or by affinity chromatography on nickel resin under denaturing conditions. Purified proteins were analyzed using liquid chromatography coupled with mass spectrometry (LC-MS/MS). Most of the E1-like activating enzymes, E2-like conjugating enzymes and the majorities of the known target as well as some previously unreported proteins for SUMO-2, SUMO-3, and NEDD8 pathways were identified.

Cell Line↗

PubMed2006

Mitochondrial matrix phosphoproteome: effect of extra mitochondrial calcium.

Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondrion-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondrial matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca(2+)) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis-mass spectrometry of Pro-Q Diamond staining, while many more Pro-Q Diamond-stained proteins evaded mass spectrometry detection. Time-dependent (32)P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins that were detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase, and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondrial matrix is extensive and dynamic. Ca(2+) has previously been shown to activate various dehydrogenases, promote the generation of reactive oxygen species (ROS), and initiate apoptosis via cytochrome c release. To evaluate the Ca(2+) signaling network, the effects of a Ca(2+) challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca(2+)-induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca(2+) dose-dependent dephosphorylation of MnSOD was associated with an approximately 2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca(2+). These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca(2+) modification of mitochondrial function at the level of MnSOD.

Animals↗

PubMed2006

High-throughput identification of IMCD proteins using LC-MS/MS.

The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.

Animals↗

PubMed2006

In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics.

We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100 mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the epsilon-amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b-ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9- to D9-trimethyl-labeled peptides of beta-casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes.

Amino Acid Sequence↗

PubMed2006

Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF.

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53 -/- cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods.

Animals↗

PubMed2006

Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes.

A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers.

Amino Acid Sequence↗

PubMed2005

Diverse range of small peptides associated with high-density lipoprotein.

High-density lipoproteins (HDL) were examined as potential carriers of small peptides in plasma. HDL purified by density gradient centrifugation was delipidated and fractionated by size-exclusion chromatography under denaturing conditions. By HPLC and mass spectrometry, more than 100 peptide components were found in the size range from 1000 to 5000 Da. By sequence analysis, peptides were identified as fragments of proteins such as apolipoproteins, fibrinogen, alpha1-proteinase inhibitor, and transthyretin. The results indicate that purified HDL bears a complex range of small peptides. It is unclear whether the peptides have any significant functional role as apolipopeptides, but they may represent a pathway for peptide delivery or scavenging and a significant reservoir of plasma peptides for diagnostic evaluation.

Apolipoproteins↗

PubMed2005

Large scale protein identification in intracellular aquaporin-2 vesicles from renal inner medullary collecting duct.

Vasopressin acts on renal collecting duct cells to stimulate translocation of aquaporin-2 (AQP2)-containing membrane vesicles from throughout the cytoplasm to the apical region. The vesicles fuse with the plasma membrane to increase water permeability. To identify the intracellular membrane compartments that contain AQP2, we carried out LC-MS/MS-based proteomic analysis of immunoisolated AQP2-containing intracellular vesicles from rat inner medullary collecting duct. Immunogold electron microscopy and immunoblotting confirmed heavy AQP2 labeling of immunoisolated vesicles. Vesicle proteins were separated by SDS-PAGE followed by in-gel trypsin digestion in consecutive gel slices and identification by LC-MS/MS. Identification of Rab GTPases 4, 5, 18, and 21 (associated with early endosomes); Rab7 (late endosomes); and Rab11 and Rab25 (recycling endosomes) indicate that a substantial fraction of intracellular AQP2 is present in endosomal compartments. In addition, several endosome-associated SNARE proteins were identified including syntaxin-7, syntaxin-12, syntaxin-13, Vti1a, vesicle-associated membrane protein 2, and vesicle-associated membrane protein 3. Rab3 was not found, however, either by mass spectrometry or immunoblotting, suggesting a relative lack of AQP2 in secretory vesicles. Additionally, we identified markers of the trans-Golgi network, components of the exocyst complex, and several motor proteins including myosin 1C, non-muscle myosins IIA and IIB, myosin VI, and myosin IXB. Beyond this, identification of multiple endoplasmic reticulum-resident proteins and ribosomal proteins indicated that a substantial fraction of intracellular AQP2 is present in rough endoplasmic reticulum. These results show that AQP2-containing vesicles are heterogeneous and that intracellular AQP2 resides chiefly in endosomes, trans-Golgi network, and rough endoplasmic reticulum.

Animals↗

PubMed2005

Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.

Amino Acid Sequence↗

PubMed2004

Identification and proteomic profiling of exosomes in human urine.

Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.

Adult↗

PubMed2004

Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: a proteomic analysis.

SUMO, a small ubiquitin-related modifier, is known to covalently attach to a number of nuclear regulatory proteins such as p53, IkappaB, promyelocytic leukemia protein and c-Jun. The sumoylation reaction is catalyzed by the SUMO protease, which exposes the C-terminal active glycine residue of the nascent SUMO, the heterodimeric SUMO activating enzyme, the SUMO conjugating enzyme, Ubc9, and SUMO protein ligases, in a manner similar to ubiquitinylation. Identification of SUMO-regulated proteins is hampered by the fact that many sumoylated proteins are present at a level below normal detection limit. This limitation was overcome by either in vivo overexpression of Myc-SUMO or in vitro sumoylation with excess biotin-SUMO and Ubc9. Sumoylated proteins so obtained were affinity purified or isolated by immunoprecipitation. The isolated sumoylated proteins were identified by sequence analysis using mass spectrometric methods. Results reveal that several heterogeneous nuclear ribonucleoproteins (hnRNPs), zinc finger proteins, and nuclear pore complex proteins were sumoylated. The sumoylation of hnRNP A1, hnRNP F, and hnRNP K were confirmed in vivo by coimmunoprecipitation. In view of the facts that hnRNPs have been implicated in RNA splicing, transport, stability, and translation, our findings suggest that sumoylation could play an important role in regulating mRNA metabolism.

Amino Acid Sequence↗

PubMed2003

Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry.

A simple and reliable method is described here for the identification and relative quantification of proteins in complex mixtures using two-dimensional liquid chromatography/tandem mass spectrometry. The method is based on the classical proteomic analysis where proteins are digested with trypsin and the resulting peptides are separated by multidimensional liquid chromatography. The separated peptides are analyzed by tandem mass spectrometry and identified via a database search algorithm such as SEQUEST. The peak areas (integrated ion counts over the peptide elution time) of all identified peptides are calculated, and the relative concentration of each protein is determined by comparing the peak areas of all peptides from that protein in one sample versus those from the other. Using this strategy, we compared the relative level of protein expression of A431 cells (an epidermal cell line) grown in the presence or absence of epidermal growth factor (EGF). Our results are consistent with the published observations of the transient effects of EGF. In addition, the difference in the concentrations of several phosphopeptides determined in our studies suggests the possibility of several new targets involved in the EGF cell-signaling pathway. This global protein identification and quantification technology should prove to be a valuable means for comparing proteomes in biological samples subjected to differential treatments.

Amino Acid Sequence↗