Quantitation Of Changes Research Articles

ITRAQ-based quantitative analysis of protein mixtures with large fold change and dynamic range

Quantitation of changes in protein abundance is key to understanding the alterations that biological systems undergo and to discovering novel biomarkers. Currently, HPLC-MS/MS can be used to quantify changes in protein expression levels [Ong, S. E. and Mann, M., Nat. Chem. Biol. 2005, 1, 252-262]. Nevertheless, quantitative analysis of protein mixtures by HPLC-MS/MS is still hampered by the wide range of protein expression levels, the high dynamic range of protein concentrations and the lack of reliable quantitation algorithms [D'Ascenzo, M., et al. Brief. Funct. Genomic. Proteomic. 2008, 7, 127-135; Lin, W. T., et al., J. Proteome Res. 2006, 5, 2328-2338; Matthiesen, R., et al. J. Proteome Res. 2005, 4, 2338-2347; Yu, C. Y., et al. Nucleic Acids Res. 2007, 35, W707-W712]. In this context, we describe two different samples (4-protmix and 8-protmix) suitable for relative protein quantitation using iTRAQ. Using the 4-protmix, relative protein changes of up to 24-fold were measured. The 8-protmix allowed the quantitation of the relative protein changes in a mixture of proteins within the range of two orders of magnitude in concentration and ten-fold differences in relative abundance. We propose that the two samples are suited to test the iTRAQ quantitative proteomic workflow. We analyzed the iTRAQ samples with a LTQ Orbitrap using "higher energy collision-induced dissociation" fragmentation [Olsen, J. V., et al., Nat. Methods 2007, 4, 709-712] and quantified with Proteome Discoverer v.1.1 (Thermo Fisher Scientific). We believe that the presented protein mixtures will be useful to assess the performance of the iTRAQ-based quantitation proteomic strategy in any laboratory.

Quantitation of Changes in VWF and FVIII Following Elective Orthopedic Surgery in Normal Individuals.

Abstract 1296Poster Board I-318 BackgroundPatients with inherited bleeding disorders undergoing surgery typically receive therapy to correct the deficient factor to a level of ∼1.0 IU/mL. There is a paucity of well-constructed descriptive data quantifying the changes in coagulation that occur in response to surgical stress in normal individuals, however. Additionally, von Willebrand factor (VWF) and factor VIII (FVIII) are acute phase reactants, and thus, supratherapeutic levels of these proteins that might develop in response to the surgical stress would predispose patients to thrombotic events. Aim: With this in mind, our objective was to quantify the changes in hemostatic variables that occur in response to elective orthopedic surgery in normal individuals. MethodsEligible subjects > 18 years of age undergoing total knee or total hip replacement were recruited. Exclusion criteria included a known bleeding disorder, hormone replacement therapy, use of oral contraceptives or pregnancy. Following informed consent, blood samples were drawn at five time points: 1) baseline - in clinic 1-2 weeks pre-operatively (pre-op), 2) pre-op the morning of surgery – same day admitting center, 3) intra-operatively (intra-op) – 30 minutes after the start of surgery, 4) 30 minutes post-operatively (post-op) - recovery room, and 5) post-op day one (POD 1) – on the ward. Samples were analyzed for complete blood counts (CBC), prothrombin time (PT), activated partial thromboplastin time (aPTT), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), FVIII coagulant activity (FVIII:C) and whole blood thromboelastography (TEG) without tissue factor activation. Repeated measures analysis of variance was used to evaluate trends over time; paired samples t-tests were done to maximize the sample size for pairwise comparisons. Results37 subjects were recruited (25 females, 12 males) with a mean age of 64 years (range 33 – 87). Means and ranges for all laboratory parameters are displayed in Table 1 in addition to statistical analysis of the change in all variables over the 5 time points. Statistically significant changes were seen in all variables except PTT and TEG angle. In general, VWF and FVIII indices decreased during surgery (presumably due to consumption) and then increased in the post-operative period (presumably due to a stress response). A great deal of variability was seen in post-operative levels of VWF and FVIII between individuals with some showing borderline low levels and some achieving very high levels. ConclusionThese data establish a baseline on which to reconsider current strategies for the peri-operative management of patients with inherited bleeding disorders, and provide relevant information when considering the relative risks of bleeding or thrombosis associated with surgery.Table 1Baseline mean (range)Pre-op mean (range)Intra-op mean (range)Post-op mean (range)POD 1 mean (range)p-value (ANOVA)*WBC X109/L (4.0 – 10.5)6.71 (4.0-10.1)6.84 (4.8-10.3)5.88 (3.3-9.5)9.31 (5.2-20.3)8.56 (5.0-13.8)<0.0001 n=20Hemoglobin g/L (120 – 160)136 (108-160)140 (119-157)120 (99-140)118 (87-146)102 (71-126)<0.0001 n=20Platelet X109/L (150 – 400)265 (160-479)273 (168-499)226 (97-477)241 (158-399)211 (124-327)<0.0001 n=20PT seconds (12.5 – 15.7)13.1 (12.3-13.8)14 (12.9-16.9)14.1 (13.2-15.4)14.5 (13.1-15.9)<0.0001 n=17PTT seconds (23 – 36)29 (25-36)29 (25-36)30 (20-39)30 (26-34)30 (26-36)0.448 n=12VWF:Ag IU/mL (0.50 – 2.00)1.46 (0.5-2.6)1.48 (0.55-2.66)1.22 (0.5-2.21)1.34 (0.54-2.19)1.72 (0.62-4.0)<0.001 n=15VWF:RCo IU/mL (0.50 – 2.00)1.39 (0.57-2.21)1.35 (0.57-2.28)1.12 (0.53-1.79)1.32 (0.49-3.23)1.65 (0.71-3.32)0.004 n=15FVIII:C IU/mL (0.50 – 1.50)1.33 (0.56-2.69)1.31 (0.62-2.91)1.01 (0.43-2.77)1.38 (0.45-4.29)1.54 (0.51-4.50)<0.0001 n=15TEG R minutes (9 – 27)8.9 (8 – 10.2)6.6 (3.7 – 8.2)0.042≠ n=4TEG angle degree (22 – 58)56 (48 – 62)57 (23 – 76)0.743≠ n=4*ANOVAs were performed on subjects with complete data sets with n= shown. Results of paired samples t-tests were generally similar (demonstrating a statistically significant difference between the baseline/intra-op, pre-op/intra-op, and intra-op/POD1 time points).≠paired samples t-tests. DisclosuresNo relevant conflicts of interest to declare.

Identification and Quantitation of Newly Synthesized Proteins in Escherichia coli by Enrichment of Azidohomoalanine-labeled Peptides with Diagonal Chromatography

A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhal-labeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively modified peptides are identified by reversed phase liquid chromatography and on-line tandem mass spectrometry. We identified 527 proteins representative of all major Gene Ontology categories. Comparing the relative amounts of 344 proteins synthesized in 15 min upon a switch of growth temperature from 37 to 44 degrees C showed that nearly 20% increased or decreased more than 2-fold. Among the most up-regulated proteins many were chaperones and proteases in accordance with the cells response to unfolded proteins due to heat stress. Comparison of our data with results from previous microarray experiments revealed the importance of regulation of gene expression at the level of transcription of the most elevated proteins under heat shock conditions and enabled identification of several candidate genes whose expression may predominantly be regulated at the level of translation. This work demonstrates for the first time the use of a bioorthogonal amino acid for proteome-wide detection of changes in the amounts of proteins synthesized during a brief period upon variations in cellular growth conditions. Comparison of such data with relative mRNA levels enables assessment of the separate contributions of transcription and translation to the regulation of gene expression.

18O Stable Isotope Labeling in MS-based Proteomics

A variety of stable isotope labeling techniques have been developed and used in mass spectrometry (MS)-based proteomics, primarily for relative quantitation of changes in protein abundances between two compared samples, but also for qualitative characterization of differentially labeled proteomes. Differential (16)O/(18)O coding relies on the (18)O exchange that takes place at the C-terminal carboxyl group of proteolytic fragments, where two (16)O atoms are typically replaced by two (18)O atoms by enzyme-catalyzed oxygen-exchange in the presence of H(2)(18)O. The resulting mass shift between differentially labeled peptide ions permits identification, characterization and quantitation of proteins from which the peptides are proteolytically generated. This review focuses on the utility of (16)O/(18)O labeling within the context of mass spectrometry-based proteome research. Different strategies employing (16)O/(18)O are examined in the context of global comparative proteome profiling, targeted subcellular proteomics, analysis of post-translational modifications and biomarker discovery. Also discussed are analytical issues related to this technique, including variable (18)O exchange along with advantages and disadvantages of (16)O/(18)O labeling in comparison with other isotope-coding techniques.

Artifactual Sulfation of Silver-stained Proteins

Sulfation and phosphorylation are post-translational modifications imparting an isobaric 80-Da addition on the side chain of serine, threonine, or tyrosine residues. These two post-translational modifications are often difficult to distinguish because of their similar MS fragmentation patterns. Targeted MS identification of these modifications in specific proteins commonly relies on their prior separation using gel electrophoresis and silver staining. In the present investigation, we report a potential pitfall in the interpretation of these modifications from silver-stained gels due to artifactual sulfation of serine, threonine, and tyrosine residues by sodium thiosulfate, a commonly used reagent that catalyzes the formation of metallic silver deposits onto proteins. Detailed MS analyses of gel-separated protein standards and Escherichia coli cell extracts indicated that several serine, threonine, and tyrosine residues were sulfated using silver staining protocols but not following Coomassie Blue staining. Sodium thiosulfate was identified as the reagent leading to this unexpected side reaction, and the degree of sulfation was correlated with increasing concentrations of thiosulfate up to 0.02%, which is typically used for silver staining. The significance of this artifact is discussed in the broader context of sulfation and phosphorylation site identification from in vivo and in vitro experiments.

A novel gallium bisaminothiolate complex as a myocardial perfusion imaging agent

The development of new myocardial perfusion imaging agents for positron emission tomography (PET) may improve the resolution and quantitation of changes in regional myocardial perfusion measurement. It is known that a (68)Ge/(68)Ga generator can provide a convenient source of PET tracers because of the long physical half-life of (68)Ge (271 days). A new ligand, 7,8-dithia-16,24-diaza-trispiro[5.2.5.2.5.3]pentacosa-15,24-diene, which consists of a N(2)S(2)-chelating core incorporated into three cyclohexyl rings, was prepared. To test feasibility and potential utility, the N(2)S(2) ligand was successfully labeled and tested with (67)Ga (half-life=3.26 day; gamma=93.3, 184.6 and 300.2 keV), which showed >92% radiochemical purity. The corresponding "cold" Ga complex was synthesized, and its structure containing a pyramidal N(2)S(2) chloride core was elucidated with X-ray crystallography. In vivo biodistribution of this novel (67)Ga complex, evaluated in normal rats, exhibited excellent heart uptake and retention, with 2.1% and 0.9% initial dose/organ at 2 and 60 min, respectively, after an intravenous injection. Autoradiography was performed in normal rats and in rats that had the left anterior descending coronary artery permanently ligated surgically. Autoradiography showed an even uptake of activity in the normal heart, and there was a distinctively lower uptake in the damaged side of the surgically modified heart. In conclusion, the new N(2)S(2) ligand was readily prepared and labeled with radioactive (67)Ga. Biodistribution in rats revealed high initial heart uptake and relatively high retention reflecting regional myocardial perfusion.

Identification and Quantitation of Changes in the Platelet Activating Factor Family of Glycerophospholipids over the Course of Neuronal Differentiation by High-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry

Glycerophospholipids are important structural lipids in membranes with changes associated with progressive neurodegenerative disorders such as Alzheimer disease. Synthesis of the platelet activating factor (PAF) glycerophospholipid subclass is implicated in the control of neuronal differentiation and death. In this article, we combine nanoflow HPLC and mass spectrometry to screen, identify, and quantitate changes in glycerophospholipid subspecies, specifically PAF family members, over the course of neuronal differentiation. Furthermore, precursor ion scans for fragments characteristic of PAF phosphocholine family members and the standard additions of PAF subspecies were combined to perform absolute quantitation of PAF lipids in undifferentiated and differentiated PC12 cells. Surprisingly, a marked asymmetry was detected in the two predominant PAF species (C16:0, C18:0) over the course of differentiation. These results describe a new technique for the sensitive analysis of lipids combining nanoflow HPLC, ESI-MS, and precursor ion scan. Limits of detection of as little as 2 pg of PAF and LPC were obtained, and analysis of the lipidome of as little as 70,000 cells was performed on this system. Furthermore, application to the PC12 model identified a quantifiable difference between PAF molecular species produced over the course of neuronal differentiation.

Quantification of sugars and sugar phosphates in Arabidopsis thaliana tissues using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry

This work reports the development and optimisation of a negative ion mode on-line LC-ESI-MS/MS method for the sensitive targeted analysis of the key glycolytic intermediates, sugars and sugar phosphates from plants, using a porous graphitic carbon (PGC) stationary phase and an MS compatible mobile phase. Using this newly developed method, separation and detection of a solution of standard compounds is achieved in less than 20 min. Target metabolite compounds were identified in plant extracts from their characteristic retention times, and product ion spectra. This on-line PGC-ESI-MS/MS method shows good linearity over the concentration range 0–100 μM, selectivity, short analysis time, and limits of detection of 0.1 μM for disaccharides trehalose (Tre), sucrose (Suc), and maltose, and 1.5 μM for hexose phosphates fructose-6-phosphate (Fru6P), glucose-1-phosphate (Glc1P), and glucose-6-phosphate (Glc6P), and phosphoenolpyruvate (PEP). This paper describes details of our method and its application to the simultaneous quantitative analysis of soluble sugars and sugar phosphates from Arabidopsis thaliana tissues. We have demonstrated the utility of our method for the analysis of biological samples by applying it to the simultaneous quantitation of changes in soluble sugars and sugar phosphates in A. thaliana Columbia-0 (Col-0) and its starchless phosphoglucomutase ( pgm) mutant over a 12-h light/12-h dark growth cycle.

8‐Plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease

An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of eight proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2- and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid (CSF) proteins from subjects undergoing intravenous Ig treatment for Alzheimer's disease. Results from this study identify a number of protein expression changes that occur in CSF after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.

A New Validation Method for X-ray Mammogram Registration Algorithms Using a Projection Model of Breast X-ray Compression

Establishing spatial correspondence between features visible in X-ray mammograms obtained at different times has great potential to aid assessment and quantitation of change in the breast indicative of malignancy. The literature contains numerous nonrigid registration algorithms developed for this purpose, but existing approaches are flawed by the assumption of inappropriate 2-D transformation models and quantitative estimation of registration accuracy is limited. In this paper, we describe a novel validation method which simulates plausible mammographic compressions of the breast using a magnetic resonance imaging (MRI) derived finite element model. By projecting the resulting known 3-D displacements into 2-D and generating pseudo-mammograms from these same compressed magnetic resonance (MR) volumes, we can generate convincing images with known 2-D displacements with which to validate a registration algorithm. We illustrate this approach by computing the accuracy for two conventional nonrigid 2-D registration algorithms applied to mammographic test images generated from three patient MR datasets. We show that the accuracy of these algorithms is close to the best achievable using a 2-D one-to-one correspondence model but that new algorithms incorporating more representative transformation models are required to achieve sufficiently accurate registrations for this application.

Tools for computational processing of LC–MS datasets: A user's perspective

Liquid chromatography–mass spectrometry (LC–MS) profiling of clinical samples for quantifying absolute ion abundances of peptides and proteins has emerged as a promising approach. Quantitation of changes in protein abundance of large number of samples is challenging and requires automatic processing means. The development of data analysis software is laborious and time-consuming. Fortunately, freely available tools have been recently introduced, which incorporate algorithms for visualization and data processing and allow the user to embed external routines for data analysis. A relevant issue related to the design and evaluation of such tools concerns usability. Properties such as easy access, large datasets management, modularity, integration with other tools, etc, are important for performing large-scale integrative data analysis with methods and visual techniques from different (possibly integrated) tools. In this paper, we consider four freely available tools recently introduced in top international journals in order to identify a list of such usability descriptors. We propose 10 descriptors that can be used both as guidelines when developing new tools and as parameters for assessing usability of existing tools. The considered tools show satisfactory usability properties, and the most recent tools exhibit improved flexibility, indicating a trend towards the design of tools that give the user a more central role in the selection, use and integration of methods and tools.

NAD metabolism and sirtuins: Metabolic regulation of protein deacetylation in stress and toxicity

Sirtuins are recently discovered NAD(+)-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins, thereby regulating the biological function of their targets. Sirtuins have been shown to increase organism and tissue survival in diverse organisms, ranging from yeast to mammals. Evidence indicates that NAD(+) metabolism and sirtuins contribute to mechanisms that influence cell survival under conditions of stress and toxicity. For example, recent work has shown that sirtuins and increased NAD(+) biosynthesis provide protection against neuron axonal degeneration initiated by genotoxicity or trauma. In light of their protective effects, sirtuins and NAD(+) metabolism could represent therapeutic targets for treatment of acute and chronic neurodegenerative conditions. Our work has focused on elucidating the enzymatic functions of sirtuins and quantifying perturbations of cellular NAD(+) metabolism. We have developed mass spectrometry methods to quantitate cellular NAD(+) and nicotinamide. These methods allow the quantitation of changes in the amounts of these metabolites in cells caused by chemical and genetic interventions. Characterization of the biochemical properties of sirtuins and investigations of NAD(+) metabolism are likely to provide new insights into mechanisms by which NAD(+) metabolism regulates sirtuin activities in cells. To develop new strategies to improve cell stress resistance, we have initiated proof of concept studies on pharmacological approaches that target sirtuins and NAD(+) metabolism, with the goal of enhancing cell protection against genotoxicity.

Preoperative weight loss with a very-low-energy diet: quantitation of changes in liver and abdominal fat by serial imaging1–3

A very-low-energy diet (VLED) can result in substantial, rapid weight loss and is increasingly prescribed before obesity surgery to minimize risk and difficulty by reducing liver size and abdominal adiposity. Despite its growing popularity, a VLED in this setting has received little attention. The aim of this study was to investigate the efficacy and acceptability of a preoperative VLED. In a prospective observational study, 32 subjects (n = 19 men and 13 women) with a mean (+/-SD) age of 47.5 +/- 8.3 y and a body mass index (in kg/m(2)) of 47.3 +/- 5.3 consumed a VLED for 12 wk. Primary outcomes included changes in liver volume (LV) and in visceral and subcutaneous adipose tissue (VAT/SAT). Changes in body weight, anthropometric measures, and biochemical variables were also recorded, and compliance with, acceptability of, and side effects of treatment were assessed. Changes in LV and VAT/SAT area were measured by computed tomography and magnetic resonance imaging at baseline and weeks 2, 4, 8, and 12. Mean (+/-SD) LV, VAT/SAT, and body weight decreased significantly (P < 0.001 for all). The degree of LV reduction was directly related to the reduction in relative body weight (r = 0.54, P = 0.001) and initial LV (r = 0.43, P = 0.015). Eighty percent of the reduction in LV occurred between weeks 0 and 2 (P < 0.001). Reductions in body weight and VAT were uniform over the 12-wk period. Attrition was 14%. Acceptability was adequate but waned over time, and mild transitory side effects occurred. Given the observed early reduction in LV and the progressive reduction in VAT, we suggest that the minimum duration for a preoperative VLED be 2 wk. Ideally, the duration should be 6 wk to achieve maximal LV reduction and significant reductions in VAT and body weight without compromising compliance and acceptability.

Preoperative weight loss with a very-low-energy diet: quantitation of changes in liver and abdominal fat by serial imaging

Preoperative weight loss with a very-low-energy diet: quantitation of changes in liver and abdominal fat by serial imaging

A Quantitative Results-driven Approach to Analyzing Multisite Protein Phosphorylation

Multisite protein phosphorylation appears to be quite common. Nevertheless our understanding of how multiple phosphorylation events regulate the function of a protein is limited in many cases. The ability to measure temporal changes in the site-specific phosphorylation profile of a protein in response to a given stimulus or cellular activity would provide an immediate indication of the functional significance of any phosphorylation site to a given process. Here we describe a mass spectrometry-based method to identify functionally relevant phosphorylation sites on a protein. It combines stable isotope labeling with a highly selective mass spectrometry analysis to detect and quantitate phosphorylation sites in response to a cellular signal. This approach requires no a priori knowledge of the phosphorylation state of the protein, does not require purification of phosphopeptides, and reliably detects substoichiometric levels of phosphorylation. Following a review of the quantitative results, only those phosphorylation sites that show a change in relative abundance are selected for identification and further study. We used this results-driven approach to study phosphorylation of the budding yeast transcription factor Pho4 in response to phosphate starvation. Phosphorylation of Pho4 on five cyclin-dependent kinase (Cdk) consensus sites has been shown to regulate the transcriptional activity of Pho4 in response to changes in environmental phosphate levels. Here we show that in phosphate-rich medium Pho4 is phosphorylated on at least 15 distinct sites including the five Cdk sites described previously. In excellent agreement with the known mechanism for regulation of Pho4 we found that phosphorylation at all five of the Cdk sites was repressed in phosphate-depleted medium. In addition to these five sites, we identified four novel phosphorylation sites that were also responsive to changes in phosphate availability. Selecting a limited number of Pho4 phosphorylation sites, we performed a more detailed kinetic analysis using an isotope-free strategy. We used LC-MS with selected reaction monitoring to greatly improve the accuracy, sensitivity, and dynamic range of the subsequent experiments. A detailed analysis of the cell-based phosphorylation at the selected Pho4 sites confirmed an apparent site preference for the Pho80-Pho85 cyclin-cyclin-dependent kinase complex.

Fluoxetine targets early progenitor cells in the adult brain

Chronic treatment with antidepressants increases neurogenesis in the adult hippocampus. This increase in the production of new neurons may be required for the behavioral effects of antidepressants. However, it is not known which class of cells within the neuronal differentiation cascade is targeted by the drugs. We have generated a reporter mouse line, which allows identification and classification of early neuronal progenitors. It also allows accurate quantitation of changes induced by neurogenic agents in these distinct subclasses of neuronal precursors. We use this line to demonstrate that the selective serotonin reuptake inhibitor antidepressant fluoxetine does not affect division of stem-like cells in the dentate gyrus but increases symmetric divisions of an early progenitor cell class. We further demonstrate that these cells are the sole class of neuronal progenitors targeted by fluoxetine in the adult brain and suggest that the fluoxetine-induced increase in new neurons arises as a result of the expansion of this cell class. This finding defines a cellular target for antidepressant drug therapies.

Calmodulin Is Required for Vasopressin-stimulated Increase in Cyclic AMP Production in Inner Medullary Collecting Duct

Calmodulin plays a critical role in regulation of renal collecting duct water permeability by vasopressin. However, specific targets for calmodulin action have not been thoroughly addressed. In the present study, we investigated whether Ca2+/calmodulin regulates adenylyl cyclase activity in the renal inner medullary collecting duct. Rat inner medullary collecting duct suspensions were incubated in the presence or absence of 0.1 nM vasopressin and the calmodulin inhibitors, monodansylcadaverine, W-7, and trifluoperazine, followed by measurement of cAMP. Vasopressin-stimulated cAMP elevation was significantly attenuated in the presence of calmodulin inhibitors. Analysis of transglutaminase 2 knock-out mice confirmed that these compounds were not acting through inhibition of transglutaminase 2 activity. Calmodulin inhibitors also blocked both cholera toxin- and forskolin-stimulated cAMP accumulation. In isolated perfused tubules, W-7 reversibly blocked vasopressin-stimulated urea permeability, a process that requires a rise in intracellular cAMP but does not appear to involve protein trafficking to the apical plasma membrane. These results suggest that calmodulin is required for vasopressin-stimulated adenylyl cyclase activity in the intact inner medullary collecting duct. Reverse transcription-PCR, immunoblotting, and immunohistochemistry revealed the presence of the calmodulin-sensitive adenylyl cyclase type 3 in the rat collecting duct, an isoform previously not known to be expressed in the collecting duct. Long-term treatment of Brattleboro rats with a vasopressin analog markedly decreased adenylyl cyclase type 3 protein abundance, providing an explanation for long-term down-regulation of vasopressin response in the collecting duct. These studies demonstrate the importance of calmodulin in the regulation of collecting duct adenylyl cyclase activity and transport function.

A new strategy for analysis of phenotype marker antigens in hollow neurospheres

Changes in expression of phenotypic markers characterizing the cells comprising intact neurospheres are difficult to determine easily and accurately. The problem is compounded by the diversity of cell populations and phenotypes involved, and consequent non-uniform protein expression across the neurosphere wall, or around the circumference. Therefore, interpreting the effects of phenotype modifying conditions has been a complex and demanding task. Here, we report a novel direct method for measuring protein expression in immunofluoerescently labeled intact neurospheres by densitometric image analysis of optical cross-sections, obtained by scanning laser confocal microscopy. To demonstrate our methodology, hollow human neurospheres were exposed to basic fibroblast growth factor (FGF2), which reportedly induces neuronal commitment in monolayer cultures of neuroprogenitor cells derived from neurospheres. We determined that this treatment downregulated nestin and vimentin, protein markers accepted to indicate an immature, uncommitted phenotype. Neuron specific enolase was only marginally affected. Our strategy allows quantitation of changes in expression of marker proteins that is comparable to Western blot analysis. In addition to discriminating heterogeneity in protein expression, suitable optics may allow the resolution down to single cell level. We propose that this novel strategy, with or without confocal microscopy, may be applied to other biological systems. Analysis of protein expression by the cells comprising tubular or cylindrical cellular structures, or approximately spherical cell aggregates, can be performed efficiently using a small sample size.

Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells.

Characterization of the surface exposed membrane subproteome of human mammary epithelial cells (strain 184 A1L5) implemented lysine specific in situ labeling of the proteins using sulfosuccinimidyl-6-(biotinamido)hexanoate, followed by enrichment of the biotinylated, tryptically digested peptides, and then liquid chromatography-tandem mass spectrometry analysis of the labeled peptides. Probing the membrane subproteome in this manner yielded unambiguous identification of proteins situated on the cell surface. The method reported can be adapted to include stable isotope labeling of proteins for quantitation of changes occurring on the cell surface in response to specific perturbations.

Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology.

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.