Hypoxia modulates A431 cellular pathways association to tumor radioresistance and enhanced migration revealed by comprehensive proteomic and functional studies.

Tumor hypoxia induces cancer cell angiogenesis, invasiveness, treatment resistance, and contributes to poor clinical outcome. However, the molecular mechanism by which tumor hypoxia exerts a coordinated effect on different molecular pathways to enhance tumor growth and survival and lead to poor clinical outcome is not fully understood. In this study, we attempt to elucidate the global protein expression and functional changes in A431 epithelial carcinoma cells induced by hypoxia and reoxygenation using iTRAQ quantitative proteomics and biochemical functional assays. Quantitative proteomics results showed that 4316 proteins were quantified with FDR<1%, in which over 1200 proteins were modulated >1.2 fold, and DNA repair, glycolysis, integrin, glycoprotein turnover, and STAT1 pathways were perturbed by hypoxia and reoxygenation-induced oxidative stress. For the first time, hypoxia was shown to up-regulate the nonhomologous end-joining pathway, which plays a central role in DNA repair of irradiated cells, thereby potentially contributing to the radioresistance of hypoxic A431 cells. The up-regulation of Ku70/Ku80 dimer, a key molecular complex in the nonhomologous end-joining pathway, was confirmed by Western blot and liquid chromatography/tandem mass spectrometry-MRM methods. Functional studies confirmed that up-regulation of glycolysis, integrin, glycoprotein synthesis, and down-regulation of STAT1 pathways during hypoxia enhanced metastastic activity of A431 cells. Migration of A431 cells was dramatically repressed by glycolysis inhibitor (2-Deoxy-d-glucose), glycoprotein synthesis inhibitor (1-Deoxynojirimycin Hydrochloride), and STAT1α overexpression that enhanced the integrin-mediated cell adhesion. These results revealed that hypoxia induced several biological processes involved in tumor migration and radioresistance and provided potential new targets for tumor therapy.

Quantitative proteomics reveals new insights into erythrocyte invasion by Plasmodium falciparum.

Differential expression of ligands in the human malaria parasite Plasmodium falciparum enables it to recognize different receptors on the erythrocyte surface, thereby providing alternative invasion pathways. Switching of invasion from using sialated to nonsialated erythrocyte receptors has been linked to the transcriptional activation of a single parasite ligand. We have used quantitative proteomics to show that in addition to this single known change, there are a significant number of changes in the expression of merozoite proteins that are regulated independent of transcription during invasion pathway switching. These results demonstrate a so far unrecognized mechanism by which the malaria parasite is able to adapt to variations in the host cell environment by post-transcriptional regulation.

Global molecular dysfunctions in gastric cancer revealed by an integrated analysis of the phosphoproteome and transcriptome.

We integrated LC-MS/MS-based and protein antibody array-based proteomics with genomics approaches to investigate the phosphoproteome and transcriptome of gastric cancer cell lines and endoscopic gastric biopsies from normal subjects and patients with benign gastritis or gastric cancer. More than 3,000 non-redundant phosphorylation sites in over 1,200 proteins were identified in gastric cancer cells. We correlated phosphoproteome data with transcriptome data sets and reported the expression of 41 protein kinases, 5 phosphatases and 65 phosphorylated mitochondrial proteins in gastric cancer cells. Transcriptional expression levels of 190 phosphorylated proteins were >2-fold higher in gastric cancer cells compared to normal stomach tissue. Pathway analysis demonstrated over-presentation of DNA damage response pathway and underscored critical roles of phosphorylated p53 in gastric cancer. This is the first study to comprehensively report the gastric cancer phosphoproteome. Integrative analysis of the phosphoproteome and transcriptome provided an expansive view of molecular signaling pathways in gastric cancer.

Quantitative proteomics discloses MET expression in mitochondria as a direct target of MET kinase inhibitor in cancer cells.

Cancer cells with MET overexpression are paradoxically more sensitive to MET inhibition than cells with baseline MET expression. The underlying molecular mechanisms are incompletely understood. Here, we have traced early responses of SNU5, a MET-overexpressing gastric cancer cell line, exposed to sublethal concentration of PHA-665752, a selective MET inhibitor, using iTRAQ-based quantitative proteomics. More than 1900 proteins were quantified, of which >800 proteins were quantified with at least five peptides. Proteins whose expression was perturbed by PHA-665752 included oxidoreductases, transfer/carrier proteins, and signaling proteins. Strikingly, 38% of proteins whose expression was confidently assessed to be perturbed by MET inhibition were mitochondrial proteins. Upon MET inhibition by a sublethal concentration of PHA-665752, mitochondrial membrane potential increased and mitochondrial permeability transition pore was inhibited concomitant with widespread changes in mitochondrial protein expression. We also showed the presence of highly activated MET in mitochondria, and striking suppression of MET activation by 50 nm PHA-665752. Taken together, our data indicate that mitochondria are a direct target of MET kinase inhibition, in addition to plasma membrane MET. Effects on activated MET in the mitochondria of cancer cells that are sensitive to MET inhibition might constitute a novel and critical noncanonical mechanism for the efficacy of MET-targeted therapeutics.

Investigation of POPX2 phosphatase functions by comparative phosphoproteomic analysis.

Identifying the substrates and biochemical pathway regulated by phosphatases has always been more challenging than finding those regulated by kinases. Here, we report the use of phosphoproteomic methods to analyse the pathways regulated by POPX2 (partner of PIX 2) phosphatase. POPX2 is a serine/threonine phosphatase, found in many cancer types. The levels of the POPX2 have been found to be up-regulated in the more invasive breast cancer cells compared with non-invasive ones. Our observations also suggest that POPX2 level is positively correlated with cell motility. Thus, finding substrates or pathways regulated by POPX2 will help to elucidate the regulatory mechanism of cancer cell motility and invasiveness. We have also developed and validated a protocol using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to enrich the phosphopeptides followed by LC-MS/MS to allow comparison between the phosphoproteomes of control and POPX2 overexpressing cells. With this approach, we were able to identify a biochemical pathway through which POPX2 exerts its apparent cellular function: the regulation of activity of glycogen synthase kinase-3, which in turn modulates extracellular signal-regulated kinase and cell motility.

Characterization of the human gastric fluid proteome reveals distinct pH-dependent protein profiles: implications for biomarker studies.

Gastric fluid is a source of gastric cancer biomarkers. However, very little is known about the normal gastric fluid proteome and its biological variations. In this study, we performed a comprehensive analysis of the human gastric fluid proteome using samples obtained from individuals with benign gastric conditions. Gastric fluid proteins were prefractionated using ultracentrifuge filters (3 kDa cutoff) and analyzed by two-dimensional gel electrophoresis (2-DE) and multidimensional LC-MS/MS. Our 2-DE analysis of 170 gastric fluid samples revealed distinct protein profiles for acidic and neutral samples, highlighting pH effects on protein composition. By 2D LC-MS/MS analysis of pooled samples, we identified 284 and 347 proteins in acidic and neutral samples respectively (FDR ≤1%), of which 265 proteins (72.4%) overlapped. However, unlike neutral samples, most proteins in acidic samples were identified from peptides in the filtrate (i.e., <3 kDa). Consistent with this finding, immunoblot analysis of six potential gastric cancer biomarkers rarely detected full-length proteins in acidic samples. These findings have important implications for biomarker studies because a majority of gastric cancer patients have neutral gastric fluid compared to noncancer controls. Consequently, sample stratification, choice of proteomic approaches, and validation strategy can profoundly affect the interpretation of biomarker findings. These observations should help to refine gastric fluid biomarker studies.

Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach.

Human monocytes' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

The quantitative proteomic response of Synechocystis sp. PCC6803 to phosphate acclimation.

BACKGROUND: Inorganic phosphate (Pi) is a critical nutrient for all life and is periodically limiting in marine and freshwater provinces, yet little is understood how organisms acclimate to fluctuations in Pi within their environment. To investigate whole cell adaptation, we grew Synechocystis sp. PCC6803, a model freshwater cyanobacterium, in 3%, and 0.3% inorganic phosphate (Pi) media. The cells were allowed to acclimate over 60 days, and cells were harvested for quantitative high throughput mass spectrometry-based proteomics using the iTRAQ™ labelling technology. RESULTS: In total, 120 proteins were identified, and 52 proteins were considered differentially abundant compared to the control. Alkaline phosphatase (APase) activities correlated significantly (p < 0.05) with observed relative PhoA abundances. PstS1 and PstS2 were both observed, yet PstS1 was not differentially more abundant than the control. Phycobilisome protein abundances appeared to be coordinated, and are significantly less abundant in 0.3% Pi than 3% Pi cultures. Also, the central metabolic cell function appears to have shifted towards the production of (NADPH) reducing energy and nucleotide sugars. CONCLUSIONS: This acclimation response bears strong similarity to the previously reported response to nitrogen deprivation within Synechocystis sp. PCC 6803. However, it also demonstrates some characteristics of desiccation stress, such as the regulation of fatty acids and increased abundance of rehydrin in the 3% Pi culture.

Identification of novel functional differences in monocyte subsets using proteomic and transcriptomic methods.

Human blood monocytes can be broadly divided into two distinct subsets: CD14+CD16- and CD14+/lowCD16+ subsets. Perturbation in their proportions in the blood has been observed in several disease conditions. Although numerous phenotypic and functional differences between the two subsets have already been described, the roles contributed by each subset during homeostasis or disease conditions are still largely unclear. To uncover novel differences to aid in elucidating their functions, we perform a global analysis of the two subsets utilizing both proteomics and transcriptomics approaches. From the proteomics and transcriptomics data, the expression of 613 genes by the two subsets is detected at both the protein and mRNA levels. These 613 genes are assessed for up-regulation in each subset at the protein and mRNA levels using a cutoff fold change of > or =|1.5| between subsets. Proteins and mRNAs up-regulated in each subset are then mapped in silico into biological functions. This mapping reveals copious functional differences between the subsets, many of which are seen at both protein and mRNA levels. For instance, expression of genes involved in F(CY) receptor-mediated phagocytosis are up-regulated in the CD14+/lowCD16+ subset, while those involved in antimicrobial function are up-regulated in the CD14+CD16- subset. We uncover novel functional differences between the monocyte subsets from differences in gene expression at the protein and mRNA levels. These functional differences would provide new insights into the different roles of the two monocyte subsets in regulating innate and adaptive immune responses.

Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation.

Coupling of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) to a sensitive linear ion trap (LTQ) mass spectrometer (MS) is a challenging, but highly promising approach for quantitative high-throughput proteomic profiling. Integration of the advantages of pulsed-Q dissociation (PQD) and collision-activated dissociation (CAD) fragmentation methods into a PQD-CAD hybrid mode, together with PQD optimization and data manipulation with a bioinformatics algorithm, resulted in a robust, sensitive and accurate iTRAQ quantitative proteomic workflow. The workflow was superior to the default PQD setting when profiling the proteome of a gastric cancer cell line, SNU5. Taken together, we established an optimized PQD-CAD hybrid workflow in LTQ-MS for iTRAQ quantitative proteomic profiling that may have wide applications in biological and biomedical research.

A comparative study of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) versus SCX-IMAC-based methods for phosphopeptide isolation/enrichment.

Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) has been introduced recently for phosphopeptide enrichment. Here we compared ERLIC with the well-established SCX-IMAC for identifying phosphopeptides in EGF-treated A431 cells. The ERLIC approach detected a higher number of phosphopeptides (17 311) than SCX-IMAC (4850), but it only detected 926 unique phosphopeptides compared to 1315 in SCX-IMAC. Only 12% unique phosphopeptides were common to both approaches, suggesting that more comprehensive phosphoproteomes could be generated by complementing SCX-IMAC with ERLIC.

Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ).

We assess the reliability of isobaric-tags for relative and absolute quantitation (iTRAQ), based on different types of replicate analyses taking into account technical, experimental, and biological variations. In total, 10 iTRAQ experiments were analyzed across three domains of life involving Saccharomyces cerevisiae KAY446, Sulfolobus solfataricus P2, and Synechocystis sp. PCC 6803. The coverage of protein expression of iTRAQ analysis increases as the variation tolerance increases. In brief, a cutoff point at +/-50% variation (+/-0.50) would yield 88% coverage in quantification based on an analysis of biological replicates. Technical replicate analysis produces a higher coverage level of 95% at a lower cutoff point of +/-30% variation. Experimental or iTRAQ variations exhibit similar behavior as biological variations, which suggest that most of the measurable deviations come from biological variations. These findings underline the importance of replicate analysis as a validation tool and benchmarking technique in protein expression analysis.

Proteomic analysis of Saccharomyces cerevisiae under high gravity fermentation conditions.

Saccharomyces cerevisiae KAY446 was utilized for ethanol production, with glucose concentrations ranging from 120 g/L (normal) to 300 g/L (high). Although grown in a high glucose environment, S. cerevisiae still retained the ability to produce ethanol with a high degree of glucose utilization. iTRAQ-mediated shotgun proteomics was applied to identify relative expression change of proteins under the different glucose conditions. A total of 413 proteins were identified from three replicate, independent LC-MS/MS runs. Unsurprisingly, many proteins in the glycolysis/gluconeogenesis pathway showed significant changes in expression level. Twenty five proteins involved in amino acid metabolism decreased their expression, while the expressions of 12 heat-shock related proteins were also identified. Under high glucose conditions, ethanol was produced as a major product. However, the assimilation of glucose as well as a number of byproducts was also enhanced. Therefore, to optimize the ethanol production under very high gravity conditions, a number of pathways will need to be deactivated, while still maintaining the correct cellular redox or osmotic state. Proteomics is demonstrated here as a tool to aid in this forward metabolic engineering.

Isobaric tags for relative and absolute quantitation (iTRAQ) reproducibility: Implication of multiple injections.

We analyzed 10 isobaric tags for relative and absolute quantitation (iTRAQ) experiments using three different model organisms across the domains of life: Saccharomyces cerevisiae KAY446, Sulfolobussolfataricus P2, and Synechocystis sp. PCC6803. A double database search strategy was employed to minimize the rate of false positives to less than 3% for all organisms. The reliability of proteins with single-peptide identification was also assessed using the search strategy, coupled with multiple analyses of samples into LC-MS/MS. The outcomes of the three LC-MS/MS analyses provided higher proteome coverage with an average increment in total proteins identified of 6%, 33%, and 50% found in S. cerevisiae, S. solfataricus, and Synechocystis sp., respectively. The iTRAQ quantification values were found to be highly reproducible across the injections, with an average coefficient of variation (CV) of 0.09 (scattering from 0.14 to 0.04) calculated based on log mean average ratio for all three organisms. Hence, we recommend multiple analyses of iTRAQ samples for greater proteome coverage and precise quantification.

Shotgun proteomics of cyanobacteria--applications of experimental and data-mining techniques.

Cyanobacteria are photosynthetic bacteria notable for their ability to produce hydrogen and a variety of interesting secondary metabolites. As a result of the growing number of completed cyanobacterial genome projects, the development of post-genomics analysis for this important group has been accelerating. DNA microarrays and classical two-dimensional gel electrophoresis (2DE) were the first technologies applied in such analyses. In many other systems, 'shotgun' proteomics employing multi-dimensional liquid chromatography and tandem mass spectrometry has proven to be a powerful tool. However, this approach has been relatively under-utilized in cyanobacteria. This study assesses progress in cyanobacterial shotgun proteomics to date, and adds a new perspective by developing a protocol for the shotgun proteomic analysis of the filamentous cyanobacterium Anabaena variabilis ATCC 29413, a model for N(2) fixation. Using approaches for enhanced protein extraction, 646 proteins were identified, which is more than double the previous results obtained using 2DE. Notably, the improved extraction method and shotgun approach resulted in a significantly higher representation of basic and hydrophobic proteins. The use of protein bioinformatics tools to further mine these shotgun data is illustrated through the application of PSORTb for localization, the grand average hydropathy (GRAVY) index for hydrophobicity, LipoP for lipoproteins and the exponentially modified protein abundance index (emPAI) for abundance. The results are compared with the most well-studied cyanobacterium, Synechocystis sp. PCC 6803. Some general issues in shotgun proteome identification and quantification are then addressed.

Comparison of protein and peptide prefractionation methods for the shotgun proteomic analysis of Synechocystis sp. PCC 6803.

Proteome analysis by gel-free "shotgun" proteomics relies on the simplification of a peptide mixture before it is analyzed in a mass spectrometer. While separation on a reverse-phase (RP) liquid chromatographic column is widely employed, a variety of other methods have been used to fractionate both proteins and peptides before this step. We compared six different protein and peptide fractionation workflows, using Synechocystis sp. PCC 6803, a useful model cyanobacterium for potential exploitation to improve its production of hydrogen and other secondary metabolites. Pre-digestion protein separation was performed by strip-based isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis, or weak anion exchange chromatography, while pre-RP peptide separation was accomplished by isoelectric focusing (IEF) or strong cation exchange chromatography. Peptides were identified using electrospray ionization quadrupole time of flight-tandem mass spectrometry. Mass spectrometry (MS) and tandem mass spectra were analyzed using ProID software employing both a single organism database and the entire NCBI non-redundant database, and a total of 776 proteins were identified using a stringent set of selection criteria. Method comparisons were made on the basis of the results obtained (number and types of proteins identified), as well as ease of use and other practical aspects. IEF-IEF protein and peptide fractionation prior to RP gave the best overall performance.