Molecular & Cellular Proteomics

Published by American Society for Biochemistry and Molecular Biology
Online ISSN: 1535-9484
Some HLA class I molecules bind a significant fraction of their constitutive peptidomes in the presence of proteasome inhibitors. In this study, A*68:01-bound peptides, and their parental proteins, were characterized through massive mass spectrometry sequencing to refine its binding motif, including the nearly exclusive preference for C-terminal basic residues. Stable isotope tagging was used to distinguish proteasome-inhibitor sensitive and resistant ligands. The latter accounted for less than 20% of the peptidome and, like in HLA-B27, arose predominantly from small and basic proteins. Under the conditions used for proteasome inhibition in vivo, epoxomicin and MG-132 incompletely inhibited the hydrolysis of fluorogenic substrates specific for the tryptic or for both the tryptic and chymotryptic subspecificities, respectively. This incomplete inhibition was also reflected in the cleavage of synthetic peptide precursors of A*68:01 ligands. For these substrates, the inhibition of the proteasome resulted in altered cleavage patterns. However these alterations did not upset the balance between cleavage at peptide bonds resulting in epitope destruction and those leading to their generation. The results indicate that inhibitor-resistant HLA class I ligands are not necessarily produced by non-proteasomal pathways. However, their generation is not simply explained by decreased epitope destruction upon incomplete proteasomal inhibition and may require additional proteolytic steps acting on incompletely processed proteasomal products.
Birdshot chorioretinopathy is a rare ocular inflammation whose genetic association with HLA-A*29:02 is the highest between a disease and a Major Histocompatibility Complex (MHC) molecule. It belongs to a group of MHC-I-associated inflammatory disorders, also including ankylosing spondylitis, psoriasis and Behcet's disease, for which endoplasmic reticulum aminopeptidases (ERAP) 1 and/or 2 have been identified as genetic risk factors. Since both enzymes are involved in the processing of MHC-I ligands, it seems reasonable that common peptide-mediated mechanisms may underlie the pathogenesis of these diseases. In this study, comparative immunopeptidomics was used to characterize >5000 A*29:02 ligands and quantify the effects of ERAP1 polymorphism and expression on the A*29:02 peptidome in human cells. The peptides predominant in an active ERAP1 context showed a higher frequency of nonamers, and bulkier amino acid side chains at multiple positions, compared to the peptides predominant in a less active ERAP1 background. Thus, ERAP1 polymorphism has a large influence, shaping the A*29:02 peptidome through length-dependent and length-independent effects. These changes resulted in increased affinity and hydrophobicity of A*29:02 ligands in an active ERAP1 context. The results reveal the nature of the functional interaction between A*29:02 and ERAP1 and suggest that this enzyme may affect the susceptibility to birdshot chorioretinopathy by altering the A*29:02 peptidome. The complexity of these alterations is such that not only peptide presentation, but also other potentially pathogenic features could be affected. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
The routine study of human malaria liver-stage biology in vitro is hampered by low infection efficiency of human hepatocellular carcinoma (HCC) lines (<0.1%), poor understanding of steady-state HCC biology, and lack of appropriate tools for trace sample analysis. HC-04 is the only HCC that supports complete development of human malaria parasites. We hypothesized that HCCs are in various intermediate stages of the epithelial-mesenchymal transition (EMT) and HC-04s retain epithelial characteristics that permit infection. We developed a facile analytical approach to test this hypothesis viz. the HC-04 response to hepatocyte growth factor (HGF). We used online two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) to quantify protein expression profiles in HC-04 pre-/post-HGF treatment and validated these results by RT-qPCR and microscopy. We successfully increased protein identification efficiency over offline-2D methods by 12-fold using less sample material, allowing robust protein quantification. We observed expected up-regulation and down-regulation of EMT protein markers in response to HGF, but also unexpected cellular responses. We also observed that HC-04 is generally more susceptible to HGF-mediated signaling than what was observed for HepG2, a widely used, but poor malaria liver stage-HCC model. Our analytical approach to understanding the basic biology of HC-04 helps us understand the factors that may influence its utility as a model for malaria liver-stage development. We observed that HC-04 treatment with HGF prior to the addition of Plasmodium falciparum sporozoites did not facilitate cell invasion, arguing for unlinking the effect of HGF on malaria liver stage development from hepatocyte invasion. Finally, our 2D-LC-MS/MS approach and broadly applicable experimental strategy should prove useful in the analysis of various hepatocyte-pathogen interactions, tumor progression and early disease events.
Schematic of the optimized K--GG sample preparation and analysis of workflow for deep coverage. Cells metabolically labeled by SILAC are lysed, and protein disulfide bonds are reduced and alkylated prior to digestion with trypsin. Peptides are separated by off-line basic RP chromatography and fractions are recombined by noncontiguous pooling. K--GG peptides are enriched using anti-Kantibody that has been chemically cross-linked to protein A-agarose beads. Enriched pools of K--GG peptides are analyzed by LC-MS/MS with statistical analysis of the data as described under "Experimental Procedures."
Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-K-ε-GG remnant antibodies. Here we describe a number of improvements to the K-ε-GG enrichment workflow including optimized antibody and peptide input requirements, antibody crosslinking, and improved offline fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of approximately 20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.
Small GTPase RAS plays a critical role in cellular signaling and oncogenic transformation. Proteomics analysis of genetically defined human ovarian cancer models identified the tumor susceptibility gene 101 (TSG101) as a downstream target of RAS oncogene. Mechanistic studies revealed a novel post-translational regulation of TSG101 through the RAS/RAF/MEK/MAPK signaling pathway and downstream molecules p14(ARF)/HDM2. Immunoanalysis using ovarian cancer samples and microtissue array revealed elevated TSG101 levels in human ovarian carcinomas. Silencing of TSG101 by short interfering RNA in ovarian cancer cells led to growth inhibition and cell death. Concurrent with the apparent growth-inhibitory effect, the levels of the CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) and hypoxia-inducible factor 1alpha (HIF-1alpha), as well as its cellular activity, were markedly reduced after TSG101 knockdown. These results demonstrate that TSG101 is important for CITED2- and HIF-1alpha-mediated cellular regulation in ovarian carcinomas.
Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (, a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.
Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.
The majority of microorganisms persist in nature as surface-attached communities often surrounded by an extracellular matrix, called biofilms. Most natural biofilms are not formed by a single species but by multiple species. Microorganisms do not only cooperate as in some multi-species biofilms but also compete about available nutrients. The Gram-negative bacterium Pseudomonas aeruginosa and the polymorphic fungus Candida albicans are two opportunistic pathogens that are often found coexisting in a human host. Several models of mixed biofilms have been reported for these organisms showing antagonistic behavior. To investigate the interaction of P. aeruginosa and C. albicans in more detail, we analyzed the secretome of single and mixed biofilms of both organisms using MALDI-TOF MS/MS at several time points. Over all 247 individual proteins were identified, 170 originated from P. aeruginosa and 77 from C. albicans. Most interestingly, P. aeruginosa in the presence of C. albicans secreted virulence factors such as exotoxin A and iron acquisition systems. In addition the high affinity iron-binding siderophore pyoverdine was identified in mixed biofilms but not in bacterial biofilms, indicating that P. aeruginosa increases its capability to sequester iron in competition with C. albicans. In contrast, C. albicans metabolism was significantly reduced, including a reduction in detectable iron acquisition proteins. The results obtained in this study show that microorganisms not only compete with the host for essential nutrients but also strongly with the present microflora in order to gain a competitive advantage. 
The receptor tyrosine kinase ErbB2 (HER-2/neu) is overexpressed in up to 30% of breast cancers and is associated with poor prognosis and an increased likelihood of metastasis especially in node-positive tumors. In this proteomic study, to identify the proteins that are associated with the aggressive phenotype of HER-2/neu-positive breast cancer, tumor cells from both HER-2/neu-positive and -negative tumors were procured by laser capture microdissection. Differentially expressed proteins in the two subsets of tumors were identified by two-dimensional electrophoresis and MALDI-TOF/TOF MS/MS. We found differential expression of several key cell cycle modulators, which were linked with increased proliferation of the HER-2/neu-overexpressing cells. Nine proteins involved in glycolysis (triose-phosphate isomerase (TPI), phosphoglycerate kinase 1 (PGK1), and enolase 1 (ENO1)), lipid synthesis (fatty acid synthase (FASN)), stress-mediated chaperonage (heat shock protein 27 (Hsp27)), and antioxidant and detoxification pathways (haptoglobin, aldo-keto reductase (AKR), glyoxalase I (GLO), and prolyl-4-hydrolase beta-isoform (P4HB)) were found to be up-regulated in HER-2/neu-positive breast tumors. HER-2/neu-dependent differential expression of PGK1, FASN, Hsp27, and GLO was further validated in four breast cancer cell lines and 12 breast tumors by immunoblotting and confirmed by partially switching off the HER-2/neu signaling in the high HER-2/neu-expressing SKBr3 cell line with Herceptin treatment. Statistical correlations of these protein expressions with HER-2/neu status were further verified by immunohistochemistry on a tissue microarray comprising 97 breast tumors. Our findings suggest that HER-2/neu signaling may result, directly or indirectly, in enhanced activation of various metabolic, stress-responsive, antioxidative, and detoxification processes within the breast tumor microenvironment. We hypothesize that these identified changes in the cellular proteome are likely to drive cell proliferation and tissue invasion and that the key cell cycle modulators involved, when uncovered by future research, would serve as naturally useful targets for the development of therapeutic strategies to negate the metastatic potential of HER-2/neu-positive breast tumors.
Lipid membranes structurally define the outer surface and internal organelles of cells. The multitude of proteins embedded in lipid bilayers are clearly functionally important, yet they remain poorly defined. Even today, integral membrane proteins represent a special challenge for current large scale shotgun proteomics methods. Here we used endothelial cell plasma membranes isolated directly from lung tissue to test the effectiveness of four different mass spectrometry-based methods, each with multiple replicate measurements, to identify membrane proteins. In doing so, we substantially expanded this membranome to 1,833 proteins, including >500 lipid-embedded proteins. The best method combined SDS-PAGE prefractionation with trypsin digestion of gel slices to generate peptides for seamless and continuous two-dimensional LC/MS/MS analysis. This three-dimensional separation method outperformed current widely used two-dimensional methods by significantly enhancing protein identifications including single and multiple pass transmembrane proteins; >30% are lipid-embedded proteins. It also profoundly improved protein coverage, sensitivity, and dynamic range of detection and substantially reduced the amount of sample and the number of replicate mass spectrometry measurements required to achieve 95% analytical completeness. Such expansion in comprehensiveness requires a trade-off in heavy instrument time but bodes well for future advancements in truly defining the ever important membranome with its potential in network-based systems analysis and the discovery of disease biomarkers and therapeutic targets. This analytical strategy can be applied to other subcellular fractions and should extend the comprehensiveness of many future organellar proteomics pursuits.
Mapping of changes in protein expression in GO categories. The green lines illustrate the connected pathways for signal transduction and G-protein regulation. Enriched categories common to both the protein expression and mRNA expression include cell-cell communication, cell adhesion, neurotransmitter secretion, and signal transduction categories as indicated with solid arrows. On the left is a separate heading showing the unique enriched categories found only in the GO analysis of protein expression data. These include mitochondrial ATPases (ATP biosynthesis) and enzymes in the tricarboxylic acid cycle.  
Interactome map of Cu,Znsuperoxide dismutase. The circles indicate the primary interactions (direct and indirect points for SOD1 (copper chaperones , p53, ciliary neurotrophic factor (CNTF), etc.). Note that many of these interactions involve only mutant SOD1 and not wt SOD1. The branches extending from the primary points show other genes that have either direct or indirect interaction with those species. New nodes for mutant SOD1 from the results of the current study are shown with the name of the affected cellular system such as neurotransmitter regulation and mitochondrial ATP generation (from Fig. 3).  
One of the causes of amyotrophic lateral sclerosis (ALS) is due to mutations in Cu,Zn-superoxide dismutase (SOD1). The mutant protein exhibits a toxic gain of function that adversely affects the function of neurons in the spinal cord, brain stem, and motor cortex. A proteomic analysis of protein expression in a widely used mouse model of ALS was undertaken to identify differences in protein expression in the spinal cords of mice expressing a mutant protein with the G93A mutation found in human ALS. Protein profiling was done on soluble and particulate fractions of spinal cord extracts using high throughput two-dimensional liquid chromatography coupled to tandem mass spectrometry. An integrated proteomics-informatics platform was used to identify relevant differences in protein expression based upon the abundance of peptides identified by database searching of mass spectrometry data. Changes in the expression of proteins associated with mitochondria were particularly prevalent in spinal cord proteins from both mutant G93A-SOD1 and wild-type SOD1 transgenic mice. G93A-SOD1 mouse spinal cord also exhibited differences in proteins associated with metabolism, protein kinase regulation, antioxidant activity, and lysosomes. Using gene ontology analysis, we found an overlap of changes in mRNA expression in presymptomatic mice (from microarray analysis) in three different gene categories. These included selected protein kinase signaling systems, ATP-driven ion transport, and neurotransmission. Therefore, alterations in selected cellular processes are detectable before symptomatic onset in ALS mouse models. However, in late stage disease, mRNA expression analysis did not reveal significant changes in mitochondrial gene expression but did reveal concordant changes in lipid metabolism, lysosomes, and the regulation of neurotransmission. Thus, concordance of proteomic and mRNA expression data within multiple categories validates the use of gene ontology analysis to compare different types of "omic" data.
Schematic overview of the experimental conditions. Control DMSO and test PCB samples were labeled with either Cy3 or Cy5, reversing the labeling for half of the samples. The internal standard corresponding to a mixture of equal amounts of control and test samples was labeled with Cy2. Four replicates were used per experimental condition.  
Final average body weight (mg) of X. laevis tadpoles (3 days posthatching) following a 3-day exposure to Aroclor 1254. Data are presented in all figures as mean (n 4) standard deviation (error bars) of the mean. Columns sharing at least one common superscript letter (a or b) are not significantly different, whereas the other differ at p 0.05. CTL, control.  
Representative 2D gels showing the protein expression profiles obtained from X. laevis tadpoles exposed for 72 h to the PCB mixture Aroclor 1254. Proteins of the samples obtained for the different experimental conditions were differentially labeled with Cy3 and Cy5. An internal standard composed of equal amounts of each sample and labeled with Cy2 was added. Labeled samples (25 g of each of the Cy3 and Cy5 labeled samples and of the Cy2 labeled internal standard) were loaded on 24-cm pH 4 –7 non-linear IPG strips and subjected to IEF. Proteins were further separated by SDS- PAGE (12.5%) in the second dimension. Arrows and numbers allocated by the DeCyder software indicate spots with significant changes in intensity (p 0.01, Student's t test in four independent gels). a, 2D gel image with proteins differentially expressed in the 0.1 ppm condition . b, 2D gel image with proteins differentially expressed in the 1 ppm condition.  
Sets of protein spots showing differences in intensity between the PBC experimental groups and the DMSO control group. The y axis represents the -fold change intensity of the protein spots where a positive value indicates an increase in abundance and a negative value indicates a decrease in abundance. Data are organized on the x axis with the down-regulated proteins on the left side and the up-regulated proteins on the right side. a, tadpoles exposed to 0.1 ppm Aroclor 1254 versus control DMSO. b, tadpoles exposed to 1 ppm Aroclor 1254 versus DMSO control group.  
Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into account to partly explain the worldwide decline of amphibians. PCBs induce deleterious effects on developing amphibians including deformities and delays in metamorphosis. However, the molecular mechanisms by which they express their toxicity during the development of tadpoles are still largely unknown. A proteomics analysis was performed on developing Xenopus laevis tadpoles exposed from 2 to 5 days postfertilization to either 0.1 or 1 ppm Aroclor 1254, a PCB mixture. Two-dimensional DIGE with a minimal labeling method coupled to nanoflow liquid chromatography-tandem mass spectrometry was used to detect and identify proteins differentially expressed under PCBs conditions. Results showed that 59 spots from the 0.1 ppm Aroclor 1254 condition and 57 spots from the 1 ppm Aroclor 1254 condition displayed a significant increase or decrease of abundance compared with the control. In total, 28 proteins were identified. The results suggest that PCBs induce mechanisms against oxidative stress (peroxiredoxins 1 and 2), adaptative changes in the energetic metabolism (enolase 1, glycerol-3-phosphate dehydrogenase, and creatine kinase muscle and brain types), and the implication of the unfolded protein response system (glucose-regulated protein, 58 kDa). They also affect, at least at the highest concentration tested, the synthesis of proteins involved in normal cytogenesis (alpha-tropomyosin, myosin heavy chain, and alpha-actin). For the first time, proteins such as aldehyde dehydrogenase 7A1, CArG binding factor-A, prolyl 4-hydroxylase beta, and nuclear matrix protein 200 were also shown to be up-regulated by PCBs in developing amphibians. These data argue that protein expression reorganization should be taken into account while estimating the toxicological hazard of wild amphibian populations exposed to PCBs.
Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of biomarkers of cancer invasion and disease aggressiveness. Although alterations in gene expression have been extensively quantified during neoplastic progression, complementary analyses of proteomic changes have been limited. Here we interrogate the proteomic alterations in a cohort of 15 prostate-derived tissues that included five each from adjacent benign prostate, clinically localized prostate cancer, and metastatic disease from distant sites. The experimental strategy couples isobaric tags for relative and absolute quantitation with multidimensional liquid phase peptide fractionation followed by tandem mass spectrometry. Over 1000 proteins were quantified across the specimens and delineated into clinically localized and metastatic prostate cancer-specific signatures. Included in these class-specific profiles were both proteins that were known to be dysregulated during prostate cancer progression and new ones defined by this study. Enrichment analysis of the prostate cancer-specific proteomic signature, to gain insight into the functional consequences of these alterations, revealed involvement of miR-128-a/b regulation during prostate cancer progression. This finding was validated using real time PCR analysis for microRNA transcript levels in an independent set of 15 clinical specimens. miR-128 levels were elevated in benign prostate epithelial cell lines compared with invasive prostate cancer cells. Knockdown of miR-128 induced invasion in benign prostate epithelial cells, whereas its overexpression attenuated invasion in prostate cancer cells. Taken together, our profiles of the proteomic alterations of prostate cancer progression revealed miR-128 as a potentially important negative regulator of prostate cancer cell invasion.
Metabolic characterization of S. pombe. A, Growth curve of the SILAC strain in EMMG and EMMS media supplemented with different concentrations of ammonium chloride. B, Growth curve of WT, SILAC, nic1, and SILACn strains in EMMG media at 25 °C. C, Scoring spindle formation defect in, plo1.ts41 cut7.24 SILAC cells during grown in the EMMG and EMMS media. Cultures were grown at 25 °C to a cell density of 1 10 6 before a shift to 37 °C at t 0 and spindle formation monitored by immunofluorescence. No bipolar spindles formed in either strain and the rate of spindle formation in each medium was identical. D, Metabolism of arginine in S. pombe. For full description of genotype of mutant strains see methods section. 
Assessment of arginine conversion using MS. A, Isotopic distribution of peptides from a triple-SILAC experiment for an arginine and a lysine containing peptide using the SILAC strain (top) and an arginine and a lysine containing peptide using the SILACn strain (bottom). B, Protein groups (blue bars) and peptide sequences (orange bars) identified in individual experiments using different isotopic versions of lysine and arginine. The lines on the top indicate the media used and the colored background indicates the strain used. 
Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Due to compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, 13C615N4-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of 13C615N4-arginine is catabolized by arginase and urease activity to 15N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We showed that disruption of Ni2+-dependent urease activity, through deletion of the sole Ni2+ transporter Nic1 in ammonium-supplemented medium, blocks this re-cycling to enable 13C615N4-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M and G2 cell cycle phases in S. pombe.
The proteome of a Medicago truncatula cell suspension culture was analyzed using two-dimensional electrophoresis and nanoscale HPLC coupled to a tandem Q-TOF mass spectrometer (QSTAR Pulsar i) to yield an extensive protein reference map. Coomassie Brilliant Blue R-250 was used to visualize more than 1661 proteins, which were excised, subjected to in-gel trypsin digestion, and analyzed using nanoscale HPLC/MS/MS. The resulting spectral data were queried against a custom legume protein database using the MASCOT search engine. A total of 1367 of the 1661 proteins were identified with high rigor, yielding an identification success rate of 83% and 907 unique protein accession numbers. Functional annotation of the M. truncatula suspension cell proteins revealed a complete tricarboxylic acid cycle, a nearly complete glycolytic pathway, a significant portion of the ubiquitin pathway with the associated proteolytic and regulatory complexes, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, chalcone, and lignin biosynthesis. Proteins were also identified from most other functional classes including primary metabolism, energy production, disease/defense, protein destination/storage, protein synthesis, transcription, cell growth/division, and signal transduction. This work represents the most extensive proteomic description of M. truncatula suspension cells to date and provides a reference map for future comparative proteomic and functional genomic studies of the response of these cells to biotic and abiotic stress.
RSV induces cellular apoptosis of N-2a cells. A, N-2a cells were treated with various concentrations of RSV (0, 20 M, 50 M, 80 M, 100 M, and 150 M) for 24 h. Chromatin condensation and fragmentation were observed by immunofluorescence staining with Hoechst 33258. Under 80 M treatment, chromatin condensed and aggregated at the nuclear membrane. B, N-2a cells were treated with various concentrations of RSV (0, 20 M, 50 M, and 80 M) for 24 h. Apoptotic cells percentage was detected by annexin V/PI staining with flow cytometry analysis. Apoptotic cells (positive for annexin V and negative for PI) were distributed in the lower and right panels. Each column was the average percentage of three independent trials and presented as mean standard deviation (*p 0.05, **p 0.01; compared against DMSO, two-tailed t test.). Compared with DMSO group, RSV treatment significantly increased the percentage of apoptotic cells. C, N-2a cells were treated with RSV as in B, and activated caspase 3 and cleaved PARP were detected by Western blotting. GAPDH was used as loading control.
Quantitative analysis of N-2a nuclear proteome after RSV treatment and GOBP analysis of the differentially expressed proteins. A, Schematic outline of the SILAC experiment. B, Volcano plot representing the p values versus intensity changes after RSV treatment. Points showing a significant (p 0.001) fold change two after RSV treatment represented the proteins differentially regulated. Red points represented for the up-regulated proteins, blue for the down-regulated protein. C, Results of GOBP enrichment analysis. The y axis shows the GOBP term. The x axis shows the -log 10 p value. p value concerns significant enrichment of the terms.
Validation of PRC2 and PRC1 components in N-2a cell after treated with RSV. A, N-2a cells were treated with 80 M RSV for 24 h. The PRC2 and PRC1 components mRNA levels were detected by qPCR. GAPDH was used as the control. All mRNA levels were not changed after RSV treatment. B, N-2a cells were treated with 80 M RSV for 24 h. The PRC2 and PRC1 components protein levels and H3K27me3 level were detected by Western blotting. H3 and GAPDH were used as control. All components protein levels and H3K27me3 level were down-regulated after RSV treatment. C, N-2a cells were treated with RSV of different concentrations (0, 20 M, 50 M, 80 M, 100 M, and 150 M) for 24 h. EZH2 level was detected by Western blotting. H3 and GAPDH were used as loading control. EZH2 was decreased in a dose-dependent manner. D, N-2a cells were treated with 80 M RSV for a series of times (0 h, 2 h, 4 h, 6 h, 12 h, and 24 h). EZH2 level was detected by Western blotting. H3 and GAPDH were used as loading control. EZH2 was decreased in a time-dependent manner. E, Cells were treated as in A. The mRNA level of CLU and NGFR were detected by qPCR analysis. GAPDH was used as the control. The bands of Western blotting were analyzed by Image J, and the relative gray-scale was presented by the column. Each column was the average ratio of three independent trials and presented as mean standard deviation (***p 0.001, **p 0.01, *p 0.05; two-tailed t test. compared with DMSO).
Both knockdown and pharmacological disruption of EZH2 induce apoptosis in N-2a cells. A, N-2a cells were transfected with EZH2 specific-siRNA or control siRNA for 48 h. The mRNA level of EZH2 was detected by qPCR. GAPDH was used as the control. Each column was the average ratio of three independent trials and presented as mean standard deviation (**p 0.01; comparison against control siRNA, two-tailed t test.). B, Cells were treated as in A. The protein levels of EZH2, H3K27me3, cleaved caspase 3, and cleaved PARP were determined by Western blotting. GAPDH was used as the loading control. C, N-2a cells were treated with 5 M DZNep for 24 h and 48 h. The mRNA levels of EZH2 were detected by qPCR. GAPDH was used as the control. Each column was the average ratio of three independent trials and presented as mean standard deviation. D, Cells were treated as in C. The protein levels of EZH2, H3K27me3, cleaved caspase 3, and cleaved PARP were determined by Western blotting. GAPDH was used as the loading control. The bands of Western blotting were analyzed by Image J, and the relative gray-scale was presented by the column. Each column was the average ratio of three independent trials and presented as mean standard deviation. (***p 0.001, **p 0.01, *p 0.05; two-tailed t test.).
MiR-137 regulates the EZH2-mediated apoptosis after RSV treatment in both N-2a cells and SH-SY5Y cells. A, Quantitation of miRNAs targeting EZH2 after RSV treatment. N-2a cells were treated with 80 M RSV for 24 h and SH-SY5Y cells with 50 M RSV for 24 h. Three miRNAs (miR-137, miR-26a, and miR-101) were examined by qPCR. U6 snRNA was an endogenous control. Each column was the average percentage of three independent trials and presented as mean standard deviation (**p 0.01; comparison against DMSO, two-tailed t test.). B, Cells were transfected with anti-miR-137 inhibitor before RSV treatment. Cell morphology was observed by optical microscopy. C, Cells were treated as in B. Apoptotic cell percentage was detected by annexin V/PI staining with flow cytometry analysis. Apoptotic cells (positive for annexin V and negative for PI) were distributed in the lower and right panels. Each column was the average percentage of three independent trials and presented as mean standard deviation. (*p 0.05, **p 0.01; comparison against RSV, two-tailed t test.) D, Cells were treated as in B. The levels of EZH2, H3K27me3, cleaved caspase 3, and cleaved PARP were determined by Western blotting. GAPDH was used as the loading control. The bands of Western blotting were analyzed by Image J, and the relative gray-scale was presented by the column. Each column was the average ratio of three independent trials and presented as mean standard deviation. (***p 0.001, **p 0.01, *p 0.05; compared with RSV, two-tailed t test.). E, Cells were treated as in B. The mRNA levels of CLU and NGFR were detected by qPCR. GAPDH was used as the control. Each column was the average ratio of three independent trials and presented as mean standard deviation. (***p 0.001, **p 0.01, *p 0.05; compared with RSV, two-tailed t test.).
Neuroblastoma is the most common pediatric extracranial solid tumor with a broad spectrum of clinical behavior and poor prognosis. Despite intensive multimodal therapy, ongoing clinical trials and basic science investigations, neuroblastoma remains a complex medical challenge with a long-term survival rate less than 40%. In our study, we found that resveratrol (3, 5, 4'-trihydroxystilbene, RSV), a naturally occurring phytoalexin, possesses an anticancer activity through blocking cell growth and inducing apoptosis in neuroblastoma cell line Neuro-2a (N-2a) cells. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative proteomic analysis, we found that 395 proteins were up-regulated and 302 proteins were down-regulated in the nucleus of N-2a cells treated with RSV. Among these, the polycomb protein histone methyltransferase EZH2 was reduced significantly, which is aberrantly overexpressed in neuroblastoma and crucial to maintain the malignant phenotype of neuroblastoma by epigenetic repression of multiple tumor suppressor genes. EZH2 reduction further led to decreased H3K27me3 level and reactivation of neuroblastoma tumor suppressor genes CLU and NGFR. Disruption EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibition with DZNep triggered cellular apoptosis in N-2a cells. We found that the up-regulation of miR-137 level was responsible for reduced EZH2 level in tumor suppression induced by RSV. Inhibition of miR-137 expression rescued the cellular apoptosis phenotypes, EZH2 reduction and CLU and NGFR reactivation, associated with RSV treatment. Taken together, our findings present for the first time, an epigenetic mechanism involving miR-137-mediated EZH2 repression in RSV-induced apoptosis and tumor suppression of neuroblastoma, which would provide a key potential therapeutic target in neuroblastoma treatment. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
Schematic representation of tobacco DRM isolation and analysis procedures. 
Classification of DRM and PM proteins according to their number of TM domains. TM domains were predicted by HMMTOP when it was not already known in the literature. 
Classification of detergent-resistant membrane proteins according to the length of their TM domains. The average number of amino acids in the TM domains was calculated on the basis of HMMTOP predictions.
Classification of DRM and PM proteins according to the GRAVY index. GRAVY values were calculated using the ProtParam tool on the ExPASy server.
Functional grouping of DRM and PM proteins. Grouping was assumed according to the putative functions given in the databases (UniProt,; and NCBI, 
A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains that play important roles in protein sorting, signal transduction, or infection by pathogens. Recent reports demonstrated the presence, in plants, of detergent-resistant fractions isolated from plasma membrane. Analysis of the lipidic composition of this fraction revealed its enrichment in sphingolipids and sterols and depletion in phospho- and glycerolipids as previously observed for animal microdomains. One-dimensional gel electrophoresis experiments indicated that these detergent-resistant fractions are able to recruit a specific set of plasma membrane proteins and exclude others. In the present study, we used mass spectrometry to give an extensive description of a tobacco plasma membrane fraction resistant to solubilization with Triton X-100. This led to the identification of 145 proteins whose functional and physicochemical characteristics were analyzed in silico. Parameters such as isoelectric point, molecular weight, number and length of transmembrane segments, or global hydrophobicity were analyzed and compared with the data available concerning plant plasma membrane proteins. Post-translational modifications, such as myristoylation, palmitoylation, or presence of a glycosylphosphatidylinositol anchor, were examined in relation to the presence of the corresponding proteins in these microdomains. From a functional point of view, this analysis indicated that if a primary function of the plasma membrane, such as transport, seems under-represented in the detergent-resistant fraction, others undergo a significant increase of their relative importance. Among these are signaling and response to biotic and abiotic stress, cellular trafficking, and cell wall metabolism. This suggests that these domains are likely to constitute, as in animal cells, signaling platforms involved in these physiological functions.
In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry and liquid chromatography matrix-assisted laser desorption/ionization tandem mass spectrometry. The use of two ionization/instrumental configurations led to complementary peptide identifications that increased the confidence of protein assignments. Examples that illustrate the power of this strategy are taken from two different projects: i) immunoaffinity purified complexes containing the prion protein from the murine brain, and ii) human tracheal epithelium gland secretions. In these studies, a large number of novel proteins were identified using stringent match criteria, in addition to many that had been identified in previous experiments. In the latter case, the ICAT method produced significant new information on changes that occur in protein expression levels in a patient suffering from cystic fibrosis.
Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [(13)C(6)]glucose incubation to evaluate the native glycated proteins labeled with [(12)C(6)]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration.
Averaged MS-spectra of one peptide from the benzene- 1,2 dioxygenase ferredoxin-NAD() reductase subunit protein (P. putida KT2440) measured by nano-LC LTQ Orbitrap-MS to demonstrate the differential isotopic pattern of 12 C peptides and 13 C-labeled peptides during the incorporation of different substrate ratios. The measured peptide with the sequence GIFAVGD- VATWPLHSGGK has a mass charge of 2 and is also listed in supplemental Table 1. At all substrate ratios, the isotopomers changed from the natural monoisotopic mass of 906.476 (A) because of the incorporation of heavy isotopes. The highest 13 C peak in the 10 atom % labeling experiment (B) with [MHH] 2 was 909.48621. The highest 13 C peak in the 25 atom % labeling experiment (C) with [MHH] 2 was 915.00487. C, The highest 13 C peak in the 25 atom % labeling experiment was [MHH] 2 915.00487. D, the highest 13 C peak in the 50 atom % labeling experiment was [MHH] 2 927.04526. E, the highest 13 C peak in the 100 atom % 13 C labeling experiment was [MHH] 2 947.61397.  
The metabolic incorporation of stable isotopes such as (13)C or (15)N into proteins has become a powerful tool for qualitative and quantitative proteome studies. We recently introduced a method that monitors heavy isotope incorporation into proteins and presented data revealing the metabolic activity of various species in a microbial consortium using this technique. To further develop our method using an liquid chromatography (LC)-mass spectrometry (MS)-based approach, we present here a novel approach for calculating the incorporation level of (13)C into peptides by using the information given in the decimal places of peptide masses obtained by modern high-resolution MS. In the present study, the applicability of this approach is demonstrated using Pseudomonas putida ML2 proteins uniformly labeled via the consumption of [(13)C(6)]benzene present in the medium at concentrations of 0, 10, 25, 50, and 100 atom %. The incorporation of (13)C was calculated on the basis of several labeled peptides derived from one band on an SDS-PAGE gel. The accuracy of the calculated incorporation level depended upon the number of peptide masses included in the analysis, and it was observed that at least 100 peptide masses were required to reduce the deviation below 4 atom %. This accuracy was comparable with calculations of incorporation based on the isotope envelope. Furthermore, this method can be extended to the calculation of the labeling efficiency for a wide range of biomolecules, including RNA and DNA. The technique will therefore allow a highly accurate determination of the carbon flux in microbial consortia with a direct approach based solely on LC-MS.
Western blotting analysis of seven different 14-3-3 isoforms in the gigantocellularis. Representative Western blots of the 14-3-3 isoforms demonstrate significant differences between SIDS cases and controls in the gigantocellularis. A representative gel is shown for each isoform analyzed. GAPDH was used as a loading control, which showed no difference in loading and abundance levels between the SIDS cases and controls. 
14-3-3 expression in different 5-HT nuclei in different populations of neurons, including 5-HT synthesizing neurons. The regional and cellular distribution of 14-3-3 expression in different neuronal subtypes and nuclei in the medulla. Letters (A–E) illustrates different 14-3-3 immunopositive neurons in different medullary nuclei. The 14-3-3 isoform is expressed in all nuclei but with variable intensities in the different neuronal subtypes. The intensity of immunostaining is heavy in the pyramidal neurons of the hypoglossal nucleus (HG) and round neurons of the principal inferior olive (PIO), moderate in the fusiform neurons of the dorsal motor nucleus of the vagus (DMX), and light in the small neurons of the nucleus of the solitary tract (NTS) and spinal trigeminal nucleus (SpV). Scale bar 100 m. Images (F–G) illustrates co-expression of 14-3-3 and TPH2 (using the PH8 antibody to label 5-HT neurons). Double-label immunofluorescent images shows the co-localization of the 14-3-3 isoform with 5-HT neurons in the (F) raphé obscurus, (ROb) and (G) gigantocellularis, (GC), both components of the medullary 5-HT system that contain 5-HT source neurons. In the merged image in the ROb, 5-HT neurons express 14-3-3 (arrowheads). In the GC, the merged image demonstrates that certain 5-HT neurons express 14-3-3 (arrowheads), but not all 5-HT neurons express 14-3-3 (asterisks), and not all 14-3-3-expressing neurons are serotonergic (cross with stroke). Scale bars 100 m. 
14-3-3 Isoform Immunostaining in a SIDS Case and a Control. Immunostaining with the 14-3-3 isoform demonstrated in the gigantocellularis from a representative SIDS cases (D; SIDS) compared with a representative control (C; CONTROL). The technical control without the primary antibody is shown for comparison (B; Negative Control). A, The location of the gigantocellularis (nGC) is shown diagrammatically for anatomic reference in the rostral medulla. The intensity of the 14-3-3 immunostaining appears visually less in the SIDS case compared with the control. 
Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42-75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MS(E)-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT(1A) receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process.
Confirmation of interactions detected by proteomic analysis of 14-3-3. A, co-immunoprecipitation of novel 14-3-3 interactors. Upper panel, HEK293T cells were transfected with plasmids encoding N-terminally HA-tagged 14-3-3 and YFP-tagged MIG-6 (left lanes in pictures of lysates and of immunoprecipitates) or as a control YFP-tagged MIG-6 alone (right lanes in pictures of lysates and of immunoprecipitates). Lower panel, transfection with plasmids encoding N-terminally HA-tagged 14-3-3 and vsv-tagged AJUBA (left lanes in pictures of lysates and of immunoprecipitates) or vsvtagged AJUBA alone (right lanes in pictures of lysates and of immunoprecipitates ). To confirm expression of the ectopic proteins, the lysates were subjected to Western blot analysis (WB) with the indicated antibodies (left panels). The right panel shows immunoprecipitations (IP) with an HA-specific antibody and subsequent detection of the (co-)precipitated proteins with the indicated antibodies. B, colocalization of MIG-6 and 14-3-3. DLD1-tTA cells were cotransfected with plasmids encoding CFP-14-3-3 and YFP-MIG-6. Pictures were taken from living cells 24 h after transfection. For detection of YFP and CFP exposure time of 100 ms was used. The right panel shows co-localization as indicated by yellow color. The left panel shows the corresponding phase-contrast representation. Magnification , 400.  
Model of the p53–14-3-3 pathway: induction of 14-3-3 as a nodal point of regulation, which mediates modulation of multiple cellular processes by protein-protein interactions. Expression of 14-3-3 is induced after DNA damage and during differentiation and senescence (1). p53 is a known mediator of the induction of 14-3-3 expression (2). In cancer cells expression of 14-3-3 is often lost due to mutation of p53 or CpG methylation. In normal cells, the induced 14-3-3 protein may affect a variety of cellular functions by associating with the ligands identified in this report. Whether 14-3-3 associates with these proteins is determined by their appropriate phosphorylation. Positive and negative regulation of the respective processes, which were inferred from the identity of the proteins identified by the TAP-MudPIT analysis (Table I), is indicated. Dotted lines are used for processes for which it has to be determined whether 14-3-3 association has an inhibitory or activating effect.  
Tandem affinity purification of 14-3-3-associated proteins . A, scheme of the 14-3-3-TAPc fusion protein used (CBP, calmodulin-binding protein; TEV, TEV protease cleavage site). B, confirmation of cellular localization of ectopic 14-3-3 proteins. DLD1-tTA cells were transfected with pBI-14-3-3-TAPc (left panel) and pBI-14-3-3-HA (right panel) and analyzed by immunofluorescence with a polyclonal rabbit antibody directed against the HA tag. A representative detection of the ectopic 14-3-3 proteins by indirect immunofluorescence (left image) and the corresponding phase-contrast picture (right image) is shown for each transfection. Magnification , 400. C, 14-3-3-TAPc interacts with proteins phosphorylated at 14-3-3 consensus binding sites. After tandem affinity purification of HEK293T cells transiently transfected with plasmids encoding the indicated proteins, detection of 14-3-3-binding proteins by Western blot analysis was performed using an antibody specific for the consensus 14-3-3-binding motif phosphorylated at the central serine residue. The two left lanes represent the initial lysates (input), and the two right lanes represent the eluates after tandem affinity purification. Purification of transfected TAPc served as a negative control. D, conditional expression of 14-3-3-TAPc and TAPc in DLD1 colorectal cancer cell lines. Stable cell lines were analyzed for inducible expression of 14-3-3-TAPc (upper panel) and TAPc (lower panel) by Western blot analysis after removal of doxycycline for 24 h (left lanes, in the presence of 100 ng/ml doxycycline; right lanes, 24 h after removal of doxycycline). 14-3-3-TAPc was detected by an anti-14-3-3 antibody . TAPc was detected by a Protein A-specific antibody. Detection of -tubulin served as a loading control.  
Ectopic 14-3-3 expression inhibits wound healing. Ectopic 14-3-3 expression was induced in DLD1-tTA-14-3-3-HA cells by removal of doxycycline for 48 h. The left panel represents cells expressing induced levels of ectopic 14-3-3-HA, and the right panel represents cells with repressed expression of 14-3-3-HA due to the presence of 100 ng/ml doxycycline. For inhibition of cell proliferation 10 g/ml mitomycin C was added 3 h before wounding of the confluent cell monolayer. Cells were monitored by time lapse microscopy . Pictures taken at identical locations at the indicated time points are shown. For details see " Experimental Procedures. "  
To comprehensively identify proteins interacting with 14-3-3 sigma in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3 sigma in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3 sigma. 25 proteins were not previously assigned to any function and were named SIP2-26 (for 14-3-3 sigma-interacting protein). Among the 92 interactors with known function were a number of proteins previously implicated in oncogenic signaling (APC, A-RAF, B-RAF, and c-RAF) and cell cycle regulation (AJUBA, c-TAK, PTOV-1, and WEE1). The largest functional classes comprised proteins involved in the regulation of cytoskeletal dynamics, polarity, adhesion, mitogenic signaling, and motility. Accordingly ectopic 14-3-3 sigma expression prevented cellular migration in a wounding assay and enhanced mitogen-activated protein kinase signaling. The functional diversity of the identified proteins indicates that induction of 14-3-3 sigma could allow p53 to affect numerous processes in addition to the previously characterized inhibitory effect on G2/M progression. The data suggest that the cancer-specific loss of 14-3-3 sigma expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes.
The 14-3-3 proteins constitute a family of abundant, highly conserved and broadly expressed acidic polypeptides that are involved in the regulation of various cellular processes such as cell-cycle progression, cell growth, differentiation, and apoptosis. One member of this family, the 14-3-3 isoform sigma, is expressed only in epithelial cells and is frequently down-regulated in a variety of human cancers. To determine the prevalence of 14-3-3 sigma silencing in bladder cancer progression, we have studied the expression of this protein in normal urothelium and bladder transitional cell carcinomas (TCCs) of various grades and stages using two-dimensional gel electrophoresis in combination with Western blotting and immunohistochemistry. We show that the expression of 14-3-3 sigma is down-regulated in invasive TCCs, particularly in lesions that are undergoing epithelial-to-mesenchymal conversion. Altered expression of 14-3-3 sigma in invasive TCCs is not due to increased externalization of the protein nor to an aberrant proliferative potential of neoplastic cells. Furthermore, we found that impaired 14-3-3 sigma expression is not associated with increased levels of the dominant-negative transcriptional regulator Delta Np63. Down-regulation of 14-3-3 sigma was confirmed by indirect immunofluorescence using a peptide-based rabbit polyclonal antibody specific for this protein. We also show that the expression of 14-3-3 sigma is highly up-regulated in pure squamous cell carcinomas. Taken together, these results provide evidence that deregulation of 14-3-3 sigma may play a key role in bladder cancer progression, in particular in differentiation events leading to epithelial-to-mesenchymal transition and stratified squamous metaplasia.
14-3-3 proteins comprise a family of highly conserved and broadly expressed multifunctional regulatory proteins that are involved in various cellular processes such as cell cycle progression, cell growth, differentiation, and apoptosis. Transcriptional expression of the sigma isoform of 14-3-3 is frequently impaired in human cancers, including carcinomas of the breast, which has led to the suggestion that this protein might be involved in the neoplastic transformation of breast epithelial cells. Here we report on the analysis of 14-3-3sigma expression in primary breast tumors using a proteomic approach complemented by immunohistochemical analysis by means of specific antibodies against this isoform. We show that the levels of expression of 14-3-3sigma were similar in non-malignant breast epithelial tissue and matched malignant tissue with only sporadic loss of expression observed in 3 of the 68 tumors examined. Moreover we show that 14-3-3sigma immunoreactivity was restricted to epithelial cells and significantly stronger in the myoepithelial cells that line the mammary ducts and lobules. The lack of expression of 14-3-3sigma in the three breast carcinomas was not associated with high levels of expression of the dominant-negative transcriptional regulator DeltaNp63 or with increased expression of estrogen-responsive finger protein, a ubiquitin-protein ligase (E3) that targets 14-3-3sigma for proteolysis. Validation of the results was performed retrospectively on an independent clinical tumor sample set using a tissue microarray containing 65 primary tumors. Our data suggest that, contrary to what was previously thought, loss of expression of 14-3-3sigma protein is not a frequent event in breast tumorigenesis.
Expression analysis of 14-3-3 proteins in transformed human epithelial amnion cells (AMA) by two-dimensional gel electrophoresis analysis . Proteins synthesized by AMA cells were separated by 2D PAGE (IEF) and visualized by silver nitrate staining. The framed area in the gel, corresponding to the portion of the gel containing the 14- 3-3 protein family, is shown enlarged in the right-hand panel of the figure. The identities of the different polypeptide spots were determined by mass spectrometric analysis and are indicated.  
Two-dimensional gel electrophoresis analysis of synchronous AMA cell cultures (A and B) and uncultured non-fractionated primary keratinocytes (C). A, expression analysis (IEF and autoradiography) of 14-3-3 proteins in total protein lysates derived from [ 35 S]methionine-labeled cultures in G 1 (2.5 and 5 h post-replating), S phase (12, 15, and 17h post-replating), G 2 (20.5 h post-replating), and M phase. B, 2D patterns (IEF and autoradiography) of [ 32 P]orthophosphate-labeled proteins from G 1 (5 h post-replating), S (15 h post-replating), and mitotic cells. C, 2D patterns (IEF) of [ 32 P]orthophosphate-labeled proteins (autoradiography; left panel) and total protein (silver nitrate staining; right panel) from primary keratinocytes. In all cases only areas of interest of the relevant gels are shown. The positions of the 14-3-3 isoforms are indicated. tm, tropomyosin 1.  
Subcellular distribution patterns of 14-3-3 in AMA cells. A, cellular distribution of 14-3-3 isoforms , , , , and in interphase cells. Yellow arrowheads indicate perinuclear localization of 14-3-3, an area of strong 14-3-3 presence showing association with the Golgi, punctate staining of 14-3-3, and membrane localization of 14-3-3, respectively. White arrowheads indicate perinuclear localization of 14-3-3 and 14-3-3. Scale bar, 10 m. B, localization of 14-3-3 isoforms during the cell cycle. Representative AMA cells at different points in the cell cycle (, , and , anaphase; , metaphase; , prometaphase) are shown for illustration. Yellow arrowheads indicate vesicular staining of 14-3-3, punctate staining of 14-3-3, and membrane localization of 14-3-3, respectively. The white arrowhead indicates perinuclear localization of 14-3-3. Scale bar, 5 m. C, increased localization of 14-3-3 to the midbody (yellow arrowheads in panels a-c) at cytokinesis. Localization to the midbody was a-specific event as no other 14-3-3 could be detected (illustrated for in panel d, yellow arrowhead). Scale bar, 2 m except in panel a (10 m). D, 143-3 does not localize to tubulin filaments during mitosis (panel a), but it is associated with centrosomes and localizes to the midbody (panel b; yellow arrowheads indicate centrosomes and the white arrowhead indicates the incipient midbody). Nuclear material was counterstained with TO-PRO-3.
Analysis of extracellular 14-3-3 proteins in AMA cells (A) and in mammary tissue (B). A, a crude cell lysate prepared from AMA cells was analyzed by IEF 2D PAGE (AMA cell lysate) and matched with the pattern of proteins present in the corresponding growth medium (externalized proteins). B, interstitial fluid extracted from mammary biopsies was analyzed by IEF 2D PAGE (externalized proteins) and matched with the pattern for the corresponding tissue lysate (mammary tissue lysate). In all cases the positions for the 14-3-3 isoforms and for some cellular proteins (cytokeratin 19, Annexin V, and Rho GDI) are indicated for reference. The identity of all indicated proteins was confirmed by MS. C, levels of expression of 14-3-3 in cultured primary HMECs. HMECs were cultured to near confluency at which point culture medium was replaced by fresh medium containing different additives (brefeldin A (BfA), prolactin (PRL), or nocodazole (Noco)). Cells and corresponding medium were collected after 4 or 24 h, and total cell lysates and corresponding medium were prepared for analysis of 14-3-3 expression by Western blot analysis. Levels of -actin were used as a normalizing factor for total amount of protein loaded. D, immunohistochemistry analysis of 14-3-3 in a breast specimen. Normal breast epithelium from a reduction mammoplasty with epithelial cells staining positive and with significant staining of myoepithelial cells (200) is shown. Apical snouts containing 14-3-3 and being shed off to the lumen of the duct (arrows) were frequently detected suggesting this to be a mechanism for externalization of 14-3-3.  
Microtubule polymerization dynamics and 14-3-3 localization . AMA wells were exposed to nocodazole (10 M) for 6 h, washed thrice with cold culture medium, and allowed to recover in drug-free medium at 37 °C. Cells were fixed at different time points (0 and 15 min; panels a or b and c, respectively) and processed for double immunofluorescence labeling using antibodies against 14- 3-3 and 14-3-3 or tubulin. Scale bar, 5 m. White arrowheads (panels a and b) indicate an increased nuclear presence of 14-3-3, whereas yellow arrowheads show localization with MTs (panel a) and with centrosome/MT-organizing centers (panels b and c).  
The 14-3-3 proteins constitute a family of highly conserved and broadly expressed multifunctional polypeptides that are involved in a variety of important cellular processes that include cell cycle progression, growth, differentiation, and apoptosis. Although the exact cellular function(s) of 14-3-3 proteins is not fully elucidated, as a rule these proteins act by binding to protein ligands, thus regulating their activity; so far more than 300 cellular proteins have been reported to interact with 14-3-3 proteins. Binding to cognate interacting partners is isoform-specific, but redundancy also exists as several binding peptides can be recognized by all isoforms, and some functions can be carried out by any isoform indistinctly. Moreover by interacting with different ligands in a spatially and temporally regulated fashion the same isoform can play multiple possibly even opposing roles where the resultant cellular outcome will be determined by the integration of the various effects. Although there is a large body of literature on specific aspects of 14-3-3 biology, not much is known on the coordinated aspects of 14-3-3 isoform expression, post-translational modifications, and subcellular localization. To address the question of isoform-specific differences, we carried out a comparative analysis of the patterns of expression, phosphorylation, and subcellular localization of the 14-3-3 beta, epsilon, sigma, tau, and zeta protein isoforms in transformed human amnion (AMA) cells. To validate as well as broaden our observations we analyzed the occurrence of the various isoforms in a large number of established cell lines and mammary and urothelial tissue specimens. Given the systematic approach we undertook and our application of isoform-discriminating technologies to the analysis of various cellular systems, we expect the data presented in this study to serve as an enabling resource for researchers working with 14-3-3 proteins.
Modern proteomic techniques have identified hundreds of proteins that bind 14-3-3s, the most widespread eukaryotic phosphoserine/threonine sensors, but accurate prediction of the target phospho-sites is difficult. Here we describe a systematic approach using synthetic peptides that tests large numbers of potential binding sites in parallel for human 14-3-3. By profiling the sequence requirements for three diverse 14-3-3 binding sites (from IRS-1, IRSp53 and GIT2), we have generated enhanced bioinformatics tools to score sites and allow more tractable testing by co-immunoprecipitation. This approach has allowed us to identify two additional sites other than Ser216 in the widely studied cell division cycle (Cdc) protein 25C, whose function depends on 14-3-3 binding. These Ser247 and Ser263 sites in human Cdc25C, which were not predicted by the existing Scansite search, are conserved across species and flank the nuclear localization region. Furthermore, we found strong interactions between 14-3-3 and peptides with the sequence Rxx[S/T]xR typical for PKC sites, and which is as abundant as the canonical Rxx[S/T]xP motif in the proteome. Two such sites are required for 14-3-3 binding in the polarity protein Numb. A recent survey of >200 reported sites identified only a handful containing this motif, suggesting that it is currently under-appreciated as a candidate binding site. This approach allows one to rapidly map 14-3-3 binding sites and has revealed alternate motifs.
The 14-3-3 interactome. A, A VisANT graph showing overlaps among the lists of proteins identified for their affinity for 14-3-3s in the proteomics screens listed in Table I, with data collated in supplemental Table S1. Each paper is assigned a node in gray and lines connect the articles to the proteins, which are assigned as green nodes. Gold standard proteins for which 14-3-3-binding sites were reported in previous low-throughput studies (1) are in red, whereas in orange are proteins for which direct binding to 14-3-3 and relevant binding sites were characterized in this study. Isoforms of kinesin heavy chain are in blue to signify that these proteins bind to 14-3-3s indirectly via phosphorylated kinesin light chains (see text). The interactive version of this graph is available under 14-3-3 partners at our laboratory web page via B, The 14-3-3 binding proteome was clustered by molecular function or cellular localization as listed in supplemental Table S3. Note that the MitoMiner database does not distinguish cytoplasmic proteins that interact with mitochondria from intrinsic mitochondrial proteins.  
Phosphorylated sites whose mutation prevents 14-3-3 binding. GFP-MFF, GFP-DBNL, ISCU-GFP, and REEP4-GFP, and the indicated single and double alanine mutants of these proteins, were immunoprecipitated on GFP-TRAP® beads from transfected cells, and tested for their ability to bind directly to 14-3-3s in Far Western assays, and by copurification with endogenous 14-3-3s from the cell extracts. For ISCU, the upper GFP signal is the intact protein (which runs as a doublet in Fig. 2) and a fainter lower band is the processed form that lacks the N-terminal signal sequence. For GFP- DBNL, the immunoprecipitated proteins were also examined for phosphorylation of Ser269 and Thr291 using phosphospecific antibodies , as characterized in supplementary Fig. S2.  
Direct phosphorylation-dependent interactions of 14-3-3s with selected proteins. Tagged recombinant proteins were isolated from lysates of transfected HEK293 cells that were grown in medium containing serum, and treated or not with calyculin A (CA) as indicated. Proteins were dephosphorylated with lambda phosphatase, or not, when the phosphatase was inhibited with EDTA, and analyzed for retention of co-purified endogenous 14-3-3 proteins (K19 pan-14-3-3 antibody). The ability of recombinant proteins to bind directly to 14-3-3s was examined by Far Western assay. DNBL, KLC2 and SMAUG2 were also analyzed by Western blotting with the indicated phospho-specific antibodies , which were characterized in supplemental Fig. S2.  
Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (, and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease.
Identification of protein-protein interactions is crucial for unraveling cellular processes and biochemical mechanisms of signal transduction. Here we describe, for the first time, the application of the tandem affinity purification (TAP) and LC-MS method to the characterization of protein complexes from transgenic mice. The TAP strategy developed in transgenic mice allows the emplacement of complexes in their physiological environment in contact with proteins that might only be specifically expressed in certain tissues while simultaneously ensuring the right stoichiometry of the TAP protein versus their binding partners and represents a novelty in proteomics approaches used so far. Mouse lines expressing TAP-tagged 14-3-3zeta protein were generated, and protein interactions were determined. 14-3-3 proteins are general regulators of cell signaling and represent up to 1% of the total brain protein. This study allowed the identification of almost 40 novel 14-3-3zeta-binding proteins. Biochemical and functional characterization of some of these interactions revealed new mechanisms of action of 14-3-3zeta in several signaling pathways, such as glutamate receptor signaling via binding to homer homolog 3 (Homer 3) and in cytoskeletal rearrangements and spine morphogenesis by binding and regulating the activity of the signaling complex formed by G protein-coupled receptor kinase-interactor 1 (GIT1) and p21-activated kinase-interacting exchange factor beta (betaPIX).
Proteins that bind to 14-3-3s in response to IGF1 in a PI 3-kinase-dependent manner. HEK293 cells cultured on 10-cm-diameter dishes in medium containing 10% (v/v) serum (labeled not serum-starved) were serum-starved for 4 h (unstimulated) and then stimulated as indicated with IGF1 at 50 ng/ml for 15 min and serum at 10% (v/v) for 15 min. Where indicated, cells were incubated with LY294002 (LY; 100 mM for 1 h) prior to stimulation. Cells were lysed in 0.3 ml/dish ice-cold lysis buffer, and 3 mg of each extract was added to 100 l of a 50% (v/v) slurry of 14-3-3-Sepharose and mixed end over end for 4 h. After washing, the protein bound to 14-3-3-Sepharose was extracted into SDS sample buffer, separated by SDS-PAGE using a 4 –12% gradient gel, and analyzed by Far-Western 14-3-3 overlay (red), which detects proteins on the blot that can bind directly to digoxigenin-labeled 14-3-3 proteins. Western blotting was also performed with the anti-PAS antibody (green).  
Experimental strategy for identifying proteins whose phosphorylation and binding to 14-3-3s is stimulated by insulin. A, as detailed under " Experimental Procedures, " HeLa suspension cell cultures were serum-starved and stimulated or not with insulin. Proteins were captured on a 14-3-3-Sepharose column, which was washed until protein was undetectable in the flow-through, and specifically bound proteins were eluted by competition with a 1 mM concentration of the 14-3-3-binding synthetic phosphopeptide ARAApSAPA. Eluates were concentrated, digested with trypsin, and incubated with formaldehyde containing no deuterium (d 0 for the preparation from insulin-stimulated cells) or two deuteriums (to give d 4 for each dimethyl group added onto the peptides from unstimulated cells). d 0 /d 4 ratios were determined as outlined under " Experimental Procedures. " B, strategy for identifying phosphopeptides derived from proteins isolated by 14-3-3 capture and release from extracts of insulin-stimulated cells. Phosphopeptides were enriched by titanium dioxide affinity as detailed further under " Experimental Procedures. "  
d 0 /d 4 (insulin) ratios of 14-3-3 affinity-purified proteins from unstimulated and insulin-stimulated cells. A, comparison of PKB/Akt and AS160 total protein levels and phosphorylation status in lysates of unstimulated and insulin-stimulated HeLa cells used for 14-3-3 phosphoproteomics screens. B, Coomassie-stained SDS gel of proteins isolated by 14-3-3 capture and release from extracts of unstimulated and insulin-stimulated cells (5 times these amounts were run on a parallel gel and used for the MS analyses). C, 14-3-3 Far-Western overlay assay of proteins isolated by 14-3-3 capture and release from extracts of unstimulated and insulin-stimulated cells. D, thumbnail of the d 0 /d 4 (insulin) rankings for 14-3-3 affinity-purified proteins where off-scale indicates the proteins that were only detected in the preparation from insulin-stimulated cells. Highlighted in pink are proteins already known to be phosphorylated and to bind to 14-3-3s in response to insulin/IGF1 as well as proteins that were discovered to be insulin/IGF1-responsive in this study. In blue are proteins that we already knew do not respond to insulin/IGF1 as well as proteins found not to be insulin/IGF1-responsive in this study (see text). E, examples of d 0 /d 4 ratio quantification for peptides from CCDC6 (d 0 /d 4 6.4 for this peptide, contributing to the overall ratio of 7.1 for three quantified peptides from this protein (Table I)) and VASP (d 0 /d 4 1.1 for this peptide with an overall ratio of 1.3 for the protein (supplemental Table 1)) showing extracted ion chromatograms after analysis in the LTQ-Orbitrap mass spectrometer.  
Phosphopeptides derived from 14-3-3-binding proteins isolated from insulin-stimulated cells. The phosphorylated sites were classified according to whether they are found within motifs that conform to RXRXX(pS/pT), RXX(pS/pT) other than RXRXX(pS/pT), (pS/pT)P, or none of these (other). One phosphorylated site that matches both RXX(pS/pT) and (pS/pT)P is included in the RXX(pS/pT) group.  
Cellular regulation of 14-3-3 binding of IRS2, CCDC6, ZNRF2, SASH1, PRAS40, VASP, and LSR. HA-IRS2, HA-CCDC6, HA-ZNRF2 (wild type and S19A mutant), HA-SASH1 (wild type and S90A mutant), HA-PRAS40, HA-VASP, and HA-LSR (wild type and S493A mutant) were isolated from transfected HEK293 cells that were stimulated as indicated, and anti-HA immunoprecipitates (IP) were analyzed by 14-3-3 overlay and Western blotting. Note that at higher exposures and in other experiments it was clear that 14-3-3 binding is not completely abolished with the ZNRF2 S19A mutation. As controls for the efficacy of stimuli and inhibitors, lysates were analyzed with antibodies against phospho-Thr 308 and phospho-Ser 473 of PKB/Akt, total PKB/Akt, phospho-Erk1/2 (pErk), and total Erk1/2. PI3, PI-103; BID, BI-D1870.  
We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d(0)/d(4)) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d(0)/d(4) values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d(0)/d(4) scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser(19) of ZNRF2 (RTRAYpS(19)GS), phospho-Ser(90) of SASH1 (RKRRVpS(90)QD), and phospho- Ser(493) of lipolysis-stimulated lipoprotein receptor (RPRARpS(493)LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.
-Novel insulin-responsive proteins identified by combined SILAC and 14-3-3 affinity analysis 
-Differential Protein-Protein interactions of the Edc3 and 14-3-3 protein complex. 
Insulin plays an essential role in metabolic homeostasis in mammals, and many of the underlying biochemical pathways are regulated via the canonical phosphatidylinositol 3-kinase/AKT pathway. To identify novel metabolic actions of insulin, we conducted a quantitative proteomics analysis of insulin-regulated 14-3-3-binding proteins in muscle cells. These studies revealed a novel role for insulin in the post-transcriptional regulation of mRNA expression. EDC3, a component of the mRNA decay and translation repression pathway associated with mRNA processing bodies, was shown to be phosphorylated by AKT downstream of insulin signaling. The major insulin-regulated site was mapped to Ser-161, and phosphorylation at this site led to increased 14-3-3 binding. Functional studies indicated that induction of 14-3-3 binding to EDC3 causes morphological changes in processing body structures, inhibition of microRNA-mediated mRNA post-transcriptional regulation, and alterations in the protein- protein interactions of EDC3. These data highlight an important new arm of the insulin signaling cascade in the regulation of mRNA utilization.
Repressive activities of various domains of RIP140 in COS-1 cells. Cells were transfected with RIP140 full-length (aa 1–1161), amino-terminal (aa 1– 495), central (aa 336 –1006), and carboxyl-terminal (aa 977–1161) domains. After 24 h, the cells were treated with a general protein deacetylase inhibitor (sodium butyrate, 10 mM) for 12 h. Luciferase reporter assays were conducted in triplicate with two repetitions in each experiment. Luciferase readings were normalized against an internal control, the lacZ (-galactosidase ) gene. The relative luciferase units due to empty vector transfection and subsequent treatment with protein deacetylase inhibitor were considered as the basal transcriptional activities, and values were regarded as 1-fold. Empty bars, hyperacetylated domains. Solid bars, untreated domains.  
Nucleocytoplasmic distribution of GFP-tagged repressive domains of RIP140 in COS-1 cells. Cells were transfected with various constructs of GFP fusion RIP140 proteins, maintained, and treated as for the reporter assay (Fig. 2). a and e, amino-terminal domain (aa 1– 495). b and f, central domain (aa 336 –1006). c and g, carboxyl-terminal domain (aa 977–1161). d and h, full-length RIP140 (aa 1–1161). a– d, control cells. e– h, protein deacetylase inhibitortreated cells. i, differential nucleocytoplasmic distribution of various repressive domains was determined by counting 200 cells expressing GFP-fused RIP140 proteins.  
Receptor-interacting protein 140 (RIP140) is a versatile co-regulator for nuclear receptors and many transcription factors and contains several autonomous repressive domains. RIP140 can be acetylated, and acetylation affects its biological activity. In this study, a comprehensive proteomic analysis using liquid chromatography-tandem mass spectroscopy was conducted to identify the in vivo acetylation sites on RIP140 purified from Sf21 insect cells. Eight acetylation sites were found within the amino-terminal and the central regions, including Lys111, Lys158, Lys287, Lys311, Lys482, Lys529, Lys607, and Lys932. Reporter assays were conducted to examine the effects of acetylation on various domains of RIP140. Green fluorescent protein-tagged fusion proteins were used to demonstrate the effect on nuclear translocation of these domains. A general inhibitor of reversible protein deacetylation was used to enrich the acetylated population of RIP140. The amino-terminal region (amino acids (aa) 1-495) was more repressive and accumulated more in the nuclei under hyperacetylated conditions, whereas hyperacetylation reduced the repressive activity and nuclear translocation of the central region (aa 336-1006). The deacetylase inhibitor had no effect on the carboxyl-terminal region (aa 977-1161) where no acetylation sites were found. Hyperacetylation also enhanced the repressive activity of the full-length protein but triggered its export into the cytosol in a small population of cells. This study revealed differential effects of post-translational modification on various domains of RIP140 through acetylation, including its effects on repressive activity and nuclear translocation of the full-length protein and its subdomains.
Characterized peptides to hemoglobin (A) and phosphorylase B (B) using LCMS E . A bar plot of the identified peptides to hemoglobin (x axis) and their corresponding average signal response (y axis) from the LCMS E analyses of each sample of the six-protein mixture is shown. The absolute quantity of hemoglobin (A) loaded onto the analytical column is indicated by the following color coding: red, 100 fmol; dark blue, 250 fmol; yellow, 500 fmol; black, 1000 fmol; green, 2500 fmol; and white, 5000 fmol. The average relative standard deviation of the mass, intensity, and retention time measurements for the characterized peptides to hemoglobin across the triplicate analyses were 1.2 ppm,  
Signal responses for peptides to the standard proteins. A 5-l injection of the following six standard proteins was analyzed by LCMS: glycogen phosphorylase B (6 pmol, 97 kDa) from rabbit, hemoglobin (10 pmol, 31 kDa total, (15 kDa) and (16 kDa)) from a cow, alcohol dehydrogenase (10 pmol, 25 kDa) from yeast, serum albumin (12.5 pmol, 70 kDa) from a cow, and enolase (15 pmol, 50 kDa) from yeast. The LCMS data were processed by PLGS, and the identified peptides were organized by decreasing intensity for each protein. The bar plots illustrate the peptides identified to alcohol dehydrogenase (A), serum albumin (B), enolase (C), hemoglobin (D), hemoglobin (E), and phosphorylase B (F). The y axis is the average intensity of the detected peptide from the triplicate analysis, and the x axis is the peptide number (sorted by descending, average intensity). The three most intense peptides for each protein are highlighted in blue. The average signal response for the three most intense peptides was calculated from the three tryptic peptides and is shown in A, B, C, D, E, and F for each of the corresponding proteins, respectively. Three ionization efficiency tiers are labeled for yeast enolase (red bar).  
Universal signal-response curve for the absolute quantification of the standard proteins. The average signal response for the three most intense tryptic peptides to each of the six proteins was obtained for all proteins found in each of the six samples. A single scatter plot of the average signal response (y axis) and the corresponding protein concentration (x axis) was produced for all the proteins in all six samples and found to be linear for over 2 orders of magnitude. The data from each of the proteins are color-coded as follows: red, yeast alcohol dehydrogenase; dark blue, bovine serum albumin; yellow, enolase; black, bovine hemoglobin; light blue, bovine hemoglobin; green, bovine / hemoglobin; and gray, rabbit phosphorylase B. A linear curve fit was calculated for the entire data set (y 27.6 (X) 7401, R 2 0.9939).  
Absolute quantity of human serum proteins from analytical (A) and biological (B) replicates. The absolute quantity of 11 well characterized human serum proteins was calculated using the described method to produce the following average concentration measurements (log 10 (pg/ml), blue circle). The average concentration values (red circle) for the human serum proteins were obtained from Specialty Laboratories and were plotted along with their expected minimum and maximum values (red whiskers). The absolute quantities obtained from  
Relative quantification methods have dominated the quantitative proteomics field. There is a need, however, to conduct absolute quantification studies to accurately model and understand the complex molecular biology that results in proteome variability among biological samples. A new method of absolute quantification of proteins is described. This method is based on the discovery of an unexpected relationship between MS signal response and protein concentration: the average MS signal response for the three most intense tryptic peptides per mole of protein is constant within a coefficient of variation of less than +/-10%. Given an internal standard, this relationship is used to calculate a universal signal response factor. The universal signal response factor (counts/mol) was shown to be the same for all proteins tested in this study. A controlled set of six exogenous proteins of varying concentrations was studied in the absence and presence of human serum. The absolute quantity of the standard proteins was determined with a relative error of less than +/-15%. The average MS signal responses of the three most intense peptides from each protein were plotted against their calculated protein concentrations, and this plot resulted in a linear relationship with an R(2) value of 0.9939. The analyses were applied to determine the absolute concentration of 11 common serum proteins, and these concentrations were then compared with known values available in the literature. Additionally within an unfractionated Escherichia coli lysate, a subset of identified proteins known to exist as functional complexes was studied. The calculated absolute quantities were used to accurately determine their stoichiometry.
Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic (15)N labeling followed by MS. Reciprocal (14)N/(15)N labeling of entire Arabidopsis thaliana plants showed that full incorporation of (15)N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and (15)N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before.
DivergentSet addresses the important but so far neglected bioinformatics task of choosing a representative set of sequences from a larger collection. We found that using a phylogenetic tree to guide the construction of divergent sets of sequences can be up to 2 orders of magnitude faster than the naive method of using a full distance matrix. By providing a user-friendly interface (available online) that integrates the tasks of finding additional sequences, building and refining the divergent set, producing random divergent sets from the same sequences, and exporting identifiers, this software facilitates a wide range of bioinformatics analyses including finding significant motifs and covariations. As an example application of DivergentSet, we demonstrate that the motifs identified by the motif-finding package MEME (Motif Elicitation by Maximum Entropy) are highly unstable with respect to the specific choice of sequences. This instability suggests that the types of sensitivity analysis enabled by DivergentSet may be widely useful for identifying the motifs of biological significance.
Representative electron micrograph of spermatogonial cell suspension from STAPUT fractions 16 –21. Spermatogonia were isolated from 9-day-old Sprague-Dawley rats. Cell preparation contained mostly type B spermatogonia whose nuclei contain dense areas of chromatin close to the nuclear membrane (B). Type A spermatogonia are larger, are lightly stained, and have a large nucleus containing fine granules of chromatin (A). To a lesser extent, cell preparation contained intermediate (In) spermatogonia that present an intermediate state in chromatin characteristics and non-germ cell contaminants (Sertoli cell fragments, peritubular cells, and Leydig cells). Bar, 10 m.  
Panoramic 2-DE images of the cytoplasmic spermatogonial proteome. The narrow range IPG gels and their corresponding regions on the broad range map are represented. Overlapping patterns are indicated. IEF, isoelectric focusing.  
Comparison of the number of individual spots detected in the narrow range gels and in the corresponding regions of the broad range gels.
Functional representation of the identified proteins.  
2-DE protein map of the cytoplasmic spermatogonial extracts. 160 g of proteins were loaded on an 18-cm IPG strip with a linear pH 5– 6 gradient for isoelectric focusing, and a 12–14% gradient SDS-polyacrylamide gel was used for SDS-PAGE. Proteins were stained with silver nitrate. Isoelectric point and molecular mass calibrations were performed using a calibration abacus (kindly provided by Amersham Biosciences) and molecular weight markers, respectively. Identified spots were assigned a number.  
Despite the essential role played by spermatogonia in testicular function, little is known about these cells. To improve our understanding of their biology, our group recently identified a set of 53 spermatogonial proteins using two-dimensional (2-D) gel electrophoresis and mass spectrometry. To continue this work, we investigated a subset of the spermatogonial proteome using narrow range immobilized pH gradients to favor the detection of less abundant proteins. A 2-D reference map of spermatogonia in the pH range 4-9 was created, and protein entities fractionated in a pH 5-6 2-D gel were further processed for protein identification. A new set of 156 polypeptides was identified by peptide mass fingerprinting and tandem mass spectrometry. These polypeptides corresponded to 102 different proteins, which reflect the complexity of post-translational modifications. Seventy-nine of these proteins were identified for the first time in spermatogonia. All identified proteins were classified into functional groups. This work represents a first step toward the establishment of a systematic spermatogonia protein database.
The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a (15)N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (K(S)) and degradation (K(D)) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify K(S) and K(D) for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. K(S) and K(D) correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive (15)N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.
This study reports the comprehensive comparison of (15)N metabolic labeling and label free proteomic strategies for quantitation, with particular focus on plant proteomics. Our investigation of proteome coverage, dynamic range and quantitative precision for a wide range of mixing ratios and protein loadings aim to aid the investigators in the decision making process during experimental design. One of the main characteristics of the label free strategy is the applicability to all starting material, which is a limitation to the metabolic labeling. However, particularly at mixing ratios up to 10-fold the (15)N metabolic labeling proved to be more precise. Contrary to usual practice based on the results from this study, we suggest that nonequal mixing ratios in metabolic labeling could further increase the proteome coverage for quantitation. On the other hand, the label free strategy, in combination with low protein loading allows the extension of the dynamic range for quantitation and it is more precise at very high ratios, which could be important for certain types of experiments.
IFI16 is degraded in a proteasome-dependent manner during HSV-1 infection. 
IFI16 co-localizes with ICP0 in discrete nuclear puncta during early stages of 
The interferon-inducible protein IFI16 has emerged as a critical antiviral factor and sensor of viral DNA. IFI16 binds nuclear viral DNA, triggering expression of antiviral cytokines during infection with herpesviruses. The knowledge of the mechanisms and protein interactions through which IFI16 exerts its antiviral functions remains limited. Here, we provide the first characterization of endogenous IFI16 interactions following infection with the prominent human pathogen herpes simplex virus 1 (HSV-1). By integrating proteomics and virology approaches, we identified and validated IFI16 interactions with both viral and host proteins that are involved in HSV-1 immunosuppressive mechanisms and host antiviral responses. We discover that during early HSV-1 infection, IFI16 is recruited to sub-nuclear puncta and subsequently targeted for degradation. We observed that the HSV-1 E3 ubiquitin ligase ICP0 is necessary, but not sufficient, for the proteasom e-mediated degradation of IFI16 following infection. We substantiate that this ICP0-mediated mechanism suppresses IFI16-dependent immune responses. Utilizing an HSV-1 strain that lacks ICP0 ubiquitin ligase activity provided a system for studying IFI16-dependent cytokine responses to HSV-1, as IFI16 levels were maintained throughout infection. We next defined temporal IFI16 interactions during this immune signaling response. We discovered and validated interactions with the viral protein ICP8 and cellular ND10 nuclear body components, sites at which HSV-1 DNA is present during infection. These interactions may be critical for IFI16 to bind to nuclear viral DNA. Altogether, our results provide critical insights into both viral inhibition of IFI16 and interactions that can contribute to IFI16 antiviral functions.
Statistical models for the analysis of protein expression changes by stable isotope labeling are still poorly developed, particularly for data obtained by 16O/18O labeling. Besides large scale test experiments to validate the null hypothesis are lacking. Although the study of mechanisms underlying biological actions promoted by vascular endothelial growth factor (VEGF) on endothelial cells is of considerable interest, quantitative proteomics studies on this subject are scarce and have been performed after exposing cells to the factor for long periods of time. In this work we present the largest quantitative proteomics study to date on the short term effects of VEGF on human umbilical vein endothelial cells by 18O/16O labeling. Current statistical models based on normality and variance homogeneity were found unsuitable to describe the null hypothesis in a large scale test experiment performed on these cells, producing false expression changes. A random effects model was developed including four different sources of variance at the spectrum-fitting, scan, peptide, and protein levels. With the new model the number of outliers at scan and peptide levels was negligible in three large scale experiments, and only one false protein expression change was observed in the test experiment among more than 1000 proteins. The new model allowed the detection of significant protein expression changes upon VEGF stimulation for 4 and 8 h. The consistency of the changes observed at 4 h was confirmed by a replica at a smaller scale and further validated by Western blot analysis of some proteins. Most of the observed changes have not been described previously and are consistent with a pattern of protein expression that dynamically changes over time following the evolution of the angiogenic response. With this statistical model the 18O labeling approach emerges as a very promising and robust alternative to perform quantitative proteomics studies at a depth of several thousand proteins.
Oral squamous cell carcinoma (OSCC) remains one of the most common cancers worldwide, and the mortality rate of this disease has increased in recent years. No molecular markers are available to assist with the early detection and therapeutic evaluation of OSCC; thus, identification of differentially expressed proteins may assist with the detection of potential disease markers and shed light on the molecular mechanisms of OSCC pathogenesis. We performed a multidimensional (16)O/(18)O proteomics analysis using an integrated ESI-ion trap and MALDI-TOF/TOF MS system and a computational data analysis pipeline to identify proteins that are differentially expressed in microdissected OSCC tumor cells relative to adjacent non-tumor epithelia. We identified 1233 unique proteins in microdissected oral squamous epithelia obtained from three pairs of OSCC specimens with a false discovery rate of <3%. Among these, 977 proteins were quantified between tumor and non-tumor cells. Our data revealed 80 dysregulated proteins (53 up-regulated and 27 down-regulated) when a 2.5-fold change was used as the threshold. Immunohistochemical staining and Western blot analyses were performed to confirm the overexpression of 12 up-regulated proteins in OSCC tissues. When the biological roles of 80 differentially expressed proteins were assessed via MetaCore analysis, the interferon (IFN) signaling pathway emerged as one of the most significantly altered pathways in OSCC. As many as 20% (10 of 53) of the up-regulated proteins belonged to the IFN-stimulated gene (ISG) family, including ubiquitin cross-reactive protein (UCRP)/ISG15. Using head-and-neck cancer tissue microarrays, we determined that UCRP is overexpressed in the majority of cheek and tongue cancers and in several cases of larynx cancer. In addition, we found that IFN-beta stimulates UCRP expression in oral cancer cells and enhances their motility in vitro. Our findings shed new light on OSCC pathogenesis and provide a basis for the future development of novel biomarkers.
Functional distribution of 938 non-redundant plasma proteins or protein groups (the complete list of identified proteins is available in Supplemental Table 1).  
LC-FTICR analysis of 1:1 labeled control plasma samples. A, a partial 2D display of the detected 18 O/ 16 O-labeled peptide pairs. The elution time is shown in a normalized scale between 0 and 1. Observed peaks (represented by spots) correspond to various eluting peptides. The heavy and light isotope-labeled pairs are easily visualized with a 4-Da mass difference. B, the ratio distribution of 891 detected peptide pairs.  
Mass spectra for selected peptide pairs. A, two different peptide pairs from C-reactive protein. B, three different peptide pairs from von Willebrand factor.  
Strategy for quantitative proteome analysis using 16 O/ 18 O labeling and the AMT tag approach. The strategy is a two-stage process. In the initial stage, a peptide AMT tag data base for plasma is generated based on extensive analyses of plasma-derived peptide samples using multidimensional LC coupled to tandem mass spectrometry. In the second stage, samples to be compared are separately labeled with 16 O and 18 O, and then the labeled samples are combined for further SCX fractionation . The fractionated, labeled samples are analyzed by high throughput LC- FTICR, and peptide pairs are identified by matching to the AMT tag data base and quantified using 18 O/ 16 O abundance ratios.  
Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.
Summary of age-and E2-related global changes in ER-protein interactions in the rat ventral hippocampus. Representation of protein spots from each group that were significantly increased (green) or decreased (red) from vehicle control samples compared with a pooled internal standard using DeCyder Analysis software. (YV, young (3-month-old) vehicle (safflower oil)-treated animals; YE, young (3-month-old) E2-treated animals; AV, aged (18-month-old) vehicle (safflower oil)-treated animals; AE, aged (18-month-old) E2-treated animals; n 3 for each group). Horizontal dimension isoelectric focusing range; vertical dimension molecular weight.
DeCyder topography, gel image analysis, and average log standard abundance of annexin V (ANXAV) in response to E2 in young and aged animals. For each panel from top left to right, 3 month: YV representative topography, YE representative topography, YV representative gel image, and YE representative gel image. 18 month: AV representative topography, AE representative topography, AV representative gel image, and AE representative gel image. Graph depicts log transformed average abundance normalized to internal standard and matched to master gel. Average calculated from three independent experiments with a biological variance of four pooled animals per experiment (n 3, BV 12). *Significance from vehicle, p 0.05.
DeCyder topography, gel image analysis, and average log standard abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in response to E2 in young and aged animals. For each panel from top left to right, 3 month: YV representative topography, YE representative topography, YV representative gel image, and YE representative gel image. 18 month: AV representative topography, AE representative topography, AV representative gel image, and AE representative gel image. Graph depicts log transformed average abundance normalized to internal standard and matched to master gel. Average calculated from three independent experiments with a biological variance of four pooled animals per experiment (n 3, BV 12). *Significance from vehicle, p 0.05.
DeCyder topography, gel image analysis, and average log standard abundance of valosin containing protein/p97 (VCP) in response to E2 in young and aged animals. For each panel from top right to left, 3 month: YV representative topography, YE representative topography, YV representative gel image, and YE representative gel image. 18 month: AV representative topography, AE representative topography, AV representative gel image, and AE representative gel image. Graph depicts log transformed average abundance normalized to internal standard and matched to master gel. Average calculated from three independent experiments with a biological variance of four pooled animals per experiment (n 3, BV 12). *Significance from vehicle, p 0.05.
A ). Our results showed that gelsolin knockdown (50 n M ) 
Recent clinical evidence suggests that the neuroprotective and beneficial effects of hormone therapy (HT) may be limited by factors of age and reproductive status. The age of patients and length of time without circulating ovarian hormones are likely to be key factors in the specific neurological outcomes of HT. However, the mechanisms underlying age-related changes in hormone efficacy have not been determined. We hypothesized that there are intrinsic changes in estrogen receptor β (ERβ) function that determine its ability to mediate the actions of 17β-estradiol (E2) in brain regions such as the ventral hippocampus. In this study, we identified and quantified a subset of ERβ protein interactions in the ventral hippocampus that were significantly altered by E2 replacement in young and aged animals, using 2 Dimensional-Differential Gel Electrophoresis (2D-DIGE), coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). This study demonstrates quantitative changes in ERβ protein:protein interactions with E2 replacement, dependent upon age in the ventral hippocampus and how these changes could alter processes such as transcriptional regulation. Thus, our data provide evidence that changes in ERβ protein interactions are a potential mechanism for age-related changes in E2 responsiveness in the brain after menopause.
Lamellar bodies (LBs) are tubulovesicular secretory organelles of epithelial cells related to lysosomes. In the epidermis, they play a crucial role in permeability barrier homeostasis, secreting their contents, lipids, a variety of hydrolases, protease inhibitors, and antimicrobial peptides, in the upper keratinocyte layers. The identification of proteins transported in epidermal LBs is still far from complete, and the way their secretion is controlled unknown. In this study, we describe the first proteomics characterization by nano-LC-MS/MS of a fraction enriched in epidermal LBs. We identified 984 proteins, including proteins known or thought to be secreted by LBs. Moreover 31 proteins corresponded to lysosomal components further suggesting that LBs are a new class of secretory lysosomes. Many of the newly found proteins could play a role in the epidermal barrier and desquamation (one acid ceramidase-like protein, apolipoproteins, glycosidases, protease inhibitors, and peptidases) and in LB trafficking (e.g. Rab, Arf, and motor complex proteins). We focus here on CLIP-170/restin, a protein that mediates interactions between organelles and microtubules. Western blotting confirmed the presence of CLIP-170 and its known effectors IQGAP1 and Cdc42 in the LB-enriched fraction. We showed, by confocal microscopy analysis of skin cryosections, that CLIP-170 was expressed in differentiated keratinocytes, first at the periphery of the nucleus then with a granular cytoplasmic labeling evocative of LBs. It was preferentially co-localized with Cdc42 and with the known LB protein cathepsin D. CLIP-170 was also largely co-localized with Rab7. This study strongly suggests a new function for CLIP-170, its involvement together with Cdc42 and/or Rab7 in the intracellular trafficking of LBs, and provides evidence that nano-LC-MS/MS combined with monodimensional electrophoresis separation constitutes a powerful method for identifying proteins in a complex mixture such as subcellular structures.
Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.
2D BN/SDS-PAGE from WCL. A, Flow diagram of WCL 2D BN/SDS-PAGE. B, Principle of 2D BN/SDS-PAGE. The hyperbolic shape of the diagonal in the second dimension is due to the gradient gel in the first dimension and a linear gel in the second. Monomeric proteins are located on the diagonal and the components of MPCs below the diagonal.  
Identification and analysis of distinct proteasomes by WCL 2D BN/SDS-PAGE. A, WCL of HEK293 cells was separated by 2D BN/SDS-PAGE (5.5-14 and 10%, respectively), and immunoblotting was performed with specific antibodies recognizing either subunits of the 20S core complex (Mcp21 and 2), or a subunit of the 19S cap of the 26S proteasome (S4 ATPase), or a subunit of the PA28 regulatory subunit (PA28). The identified MPCs are shown schematically below the corresponding spots on the blot. B, An identical sample was boiled in 1% SDS, resolved by 2D BN/SDS-PAGE, immunoblotted, and probed as described in A. C, The immuno-proteasome was induced in HeLa cells by IFN-treatment for 3 or 5 days. WCL were prepared and resolved by 2D BN/SDS-PAGE. The corresponding membrane was probed with antibodies against LMP2 (upper three panels), a specific subunit of the immunoproteasome, and against the household proteasome subunits 2 and Mcp21 (lower panel). The faint spots marked with an asterisk in A and C indicate residual signal from the previous antibody probings (anti-2/Mcp21 and anti-LMP2, respectively). D, Antibody shift of Mcp21 containing protein complexes. The dialyzed HEK293 WCL was incubated with an anti-Mcp21 antibody before separation by WCL BN/SDS-PAGE and immunoblotted with the indicated antibodies.
Identification of MPCs by immunoblotting. HEK293 or J558Lm/mb1 (for the BAP proteins) WCLs were separated by 2D BN/SDS-PAGE (5.5–14 and 10%, respectively) and immunoblotted using antibodies against the indicated proteins.  
Visualization of MPCs on a 2D WCL BN/SDS gel and identification of MPC components by MS. A, WCL of HEK293 cells was prepared using Triton X-100 and separated by 2D BN/SDS-PAGE (5.5–17 and 10%, respectively). Corresponding spots A–J were isolated from a similar gel in which a larger quantity of WCL had been loaded and the proteins identified by LC-MS/MS. B, WCL of HEK293 cells was boiled with 1% SDS before separation and staining. The area between the dotted lines (diagonal) shows the migration of monomeric proteins.  
Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after gamma interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics.
Immunohistochemical analysis of Op18. A, cytoplasmic staining of Op18 in a moderately differentiated adenocarcinoma (arrow ). Original magnification, 400. B, low levels of Op18 staining in normal lung tissue; higher staining in pulmonary macrophages (arrow ). Original magnification, 200.  
The comparison of the average expression value for Op18, Op18a, and PCNA proteins in A549 and SKLU1 cell lines. Op18 and Op18a levels were higher in SKLU1 than in A549, and PCNA was higher in A549 than in SKLU1.  
Effect of DX IL-6 on Op18 and Op18a expression in A549 and SKLU1 cell lines. Op18 was decreased following DX IL-6 treatment for 24-72 h as compared with controls (0 h) in both A549 and SKLU1 cell lines (A), but Op18a increased after DX IL-6 treatment for 72 h in A549 cells and at 48 h in SKLU1 cells (B). Exp/con represents experiment/control.
We examined the expression of oncoprotein 18 (Op18) in 93 lung adenocarcinomas and 10 uninvolved lung samples using quantitative two-dimensional PAGE analysis with confirmation by mass spectrometry and two-dimensional Western blot analysis. mRNA expression was examined using oligonucleotide microarrays, and the cellular localization of the Op18 protein was examined using immunohistochemical analysis of tissue microarrays. Three phosphorylated forms and one unphosphorylated form of the Op18 protein were identified and found to be overexpressed in lung adenocarcinomas as compared with normal lung. The percentage of phosphorylated to total Op18 protein isoforms increased from 3.2% in normal lung to 7.9% in lung tumors. Both the phosphorylated and unphosphorylated Op18 proteins were significantly increased in poorly differentiated tumors as compared with moderately or well differentiated lung adenocarcinomas (p<0.03), suggesting that up-regulated expression of Op18 reflects a poor differentiation status and higher cell proliferation rates. This was further verified in A549 and SKLU1 lung adenocarcinoma cell lines by examining Op18 levels and phosphorylation status following treatment that altered either cell proliferation or differentiation. The increased expression of Op18 protein was significantly correlated with its mRNA level indicating that increased transcription likely underlies elevated expression of Op18. The overexpression of Op18 proteins in poorly differentiated lung adenocarcinomas and the elevated expression of the phosphorylated forms of Op18 may offer a new target for drug- or gene-directed therapy and may have potential utility as a tumor marker.
The use of 18 O-labeled proteome reference as GIS for SRM quantification. A, overall conceptual workflow. A reference sample is generated by pooling aliquots from digested individual biological samples and 18 O-labeled. The labeled proteome reference as GIS is spiked into each sample prior to LC-SRM-MS analysis. B, a workflow for proof of concept experiments.  
Calibration curves of selected peptides from standard proteins. Calibration curves of two peptides from equine skeletal muscle myoglobin in both natural and log scale: LFTGHPETLEK 3 (A) and VEADIAGHGQEVLIR 3 (B). The precision of quantification for both 16 O/ 18 O ion-pair (blue error bars) and label-free approach (red error bars) is shown. The error bar means standard deviation. A smaller error bar (marked as an asterisk in B) of label-free approach in the high end is due to the saturated peak intensity of the unlabeled transition. 95% confidence intervals for the slope and y intercept are indicated in the linear equations derived from the ion-pair data. The concentration ranges of all samples are included in Table I.  
Accurate relative quantification of human plasma proteins. The detected peak area ratios accurately match with the known concentration ratios. 95% confidence intervals for the slope and y intercept are indicated in the linear equations. The four panels represent four selected proteins.  
18 O heavy isotope incorporation . 18 O-Labeled VLVLDTDYKK 2 from -lactoglobulin detected by LTQ-Orbitrap and triple quadrupole instrument. A, LTQ-Orbitrap MS spectrum. B, extracted ion chromatogram of fully labeled ( 18 O 2 ), singly labeled ( 18 O 1 ), and unlabeled ( 16 O 2 ) peak from LTQ-Orbitrap . C, SRM extracted ion chromatogram of fully 18 O 2 -labeled transitions (solid lines) and light unlabeled transitions (dashed lines in zoom-in view) from LC-TSQ. Green, y6; blue, y7; red, y8 ion, respectively. The intensities of the light transitions are near the noise level.  
Effect of Q1 window resolution on 16 O/ 18 O area ratio. The 16 O/ 18 O area ratios of four peptides are relatively consistent with 7% variations despite the increases of Q1 resolution from 0.5 to 3.0 Da.  
Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.
16O/18O labeling is one differential proteomics technology among many that promises diagnostic and prognostic biomarkers of disease. Although the incorporation of 18O in the C-terminal carboxyl group during endoproteinase digestion in the presence of H2 18O makes the process of labeling facile, the ease and effectiveness of label incorporation have in some regards been outweighed by the difficulties in interpreting the resulting spectra. Complex isotope patterns result from the composition of unlabeled (18O(0)), singly labeled (18O(1)), and doubly labeled species (18O(2)) as well as contributions from the naturally occurring isotopes (e.g. 13C and 15N). Moreover because labeling is enzymatic, the number of 18O atoms incorporated can vary from peptide to peptide. Finally it is difficult to distinguish highly up-regulated from highly down-regulated or C-terminal peptides. We have developed an algorithm entitled regression analysis applied to mass spectrometry (RAAMS) that automatically, rapidly, and confidently interprets spectra of 18O-labeled peptides without requiring chemical composition information derived from product ion spectra. The algorithm is able to measure the effective 18O incorporation rate due to variable enzyme substrate specificity of the pseudosubstrate during the isotope exchange reaction and corrects for the 18O(0) abundance that remains in the labeled sample when using a two-step digestion/labeling procedure. We have also incorporated a method for distinguishing pure 18O(0) from pure 18O(2) peptides utilizing impure H2 18O. The algorithm operates on centroided peak lists and is therefore very fast: nine chromatograms of, on average, 1,168 spectra and containing, on average, 6,761 isotopic clusters were interpreted in, on average, 45 s per chromatogram. RAAMS is fast enough (average, 38 ms/spectrum) to allow the possibility of performing information-dependent MS/MS on a chromatographic time scale on species exceeding predetermined ratio thresholds. We describe in detail the operation of the algorithm and demonstrate its use on datasets with known and unknown ratios.
Quantitative strategies relying on stable isotope labeling and isotope dilution mass spectrometry have proven to be a very robust alternative to the well established gel-based techniques for the study of the dynamic proteome. Postdigestion 18O labeling is becoming very popular mainly due to the simplicity of the enzyme-catalyzed exchange reaction, the peptide handling and storage procedures, and the flexibility and versatility introduced by decoupling protein digestion from peptide labeling. Despite recent progresses, peptide quantification by postdigestion 18O labeling still involves several computational problems. In this work we analyzed the behavior of large collections of peptides when they were subjected to postdigestion labeling and concluded that this process can be explained by a universal kinetic model. On the basis of this observation, we developed an advanced quantification algorithm for this kind of labeling. Our method fits the entire isotopic envelope to parameters related with the kinetic exchange model, allowing at the same time an accurate calculation of the relative proportion of peptides in the original samples and of the specific labeling efficiency of each one of the peptides. We demonstrated that the new method eliminates artifacts produced by incomplete oxygen exchange in subsets of peptides that have a relatively low labeling efficiency and that may be considered indicative of false protein ratio deviations. Finally using a rigorous statistical analysis based on the calculation of error rates associated with false expression changes, we showed the validity of the method in the practice by detecting significant expression changes, produced by the activation of a model preparation of T cells, with only 5 microg of protein in three proteins among a pool of more than 100. By allowing a full control over potential artifacts, our method may improve automation of the procedures for relative protein quantification using this labeling strategy.
Top-cited authors
Juergen Cox
  • Max Planck Institute of Biochemistry
Mathias Uhlen
  • KTH Royal Institute of Technology
Steven Carr
  • Broad Institute of MIT and Harvard
Emma Lundberg
  • KTH Royal Institute of Technology
Eric W Deutsch
  • Institute for Systems Biology