Molecular & Cellular Proteomics

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.

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 (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 (http://mint.bio.uniroma2.it/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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.