Strategies for the enrichment and identification of basic proteins in proteome projects

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) - tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.     

Analysis of the grasspea proteome and identification of stress-responsive proteins upon exposure to high salinity, low temperature, and abscisic acid treatment

Abiotic stress causes diverse biochemical and physiological changes in plants and limits crop productivity. Plants respond and adapt to such stress by altering their cellular metabolism and activating various defense machineries. To understand the molecular basis of stress tolerance in plants, we have developed differential proteomes in a hardy legume, grasspea (Lathyrus sativus L.). Five-week-old grasspea seedlings were subjected independently to high salinity, low temperature and abscisic acid treatment for duration of 36 h. The physiological changes of stressed seedlings were monitored, and correlated with the temporal changes of proteome using two-dimensional gel electrophoresis. Approximately, 400 protein spots were detected in each of the stress proteome with one-fourth showing more than 2-fold differences in expression values. Eighty such proteins were subjected to LC-tandem MS/MS analyses that led to the identification of 48 stress-responsive proteins (SRPs) presumably involved in a variety of functions, including metabolism, signal transduction, protein biogenesis and degradation, and cell defense and rescue. While 33 proteins were responsive to all three treatments, 15 proteins were expressed in stress-specific manner. Further, we explored the possible role of ROS in triggering the stress-induced degradation of large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco). These results might help in understanding the spectrum of stress-regulated proteins and the biological processes they control as well as having implications for strategies to improve stress adaptation in plants.     

Evaluation of ubiquitinated proteins by proteomics reveals the role of the ubiquitin proteasome system in the regulation of Grp75 and Grp78 chaperone proteins during intestinal inflammation

The ubiquitin proteasome system (UPS) is the major pathway of intracellular protein degradation and may be involved in the pathophysiology of inflammatory bowel diseases or irritable bowel syndrome. UPS specifically degrades proteins tagged with an ubiquitin chain. We aimed to identify polyubiquitinated proteins during inflammatory response in intestinal epithelial HCT‐8 cells by a proteomic approach. HCT‐8 cells were incubated with interleukin 1β, tumor necrosis factor‐α, and interferon‐γ for 2 h. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using antiubiquitin antibody. Differential ubiquitinated proteins were then identified by LC‐ESI MS/MS. Seven proteins were differentially ubiquitinated between control and inflammatory conditions. Three of them were chaperones: Grp75 and Hsc70 were more ubiquitinated (p < 0.05) and Grp78 was less ubiquitinated (p < 0.05) under inflammatory conditions. The results for Grp75 and Grp78 were then confirmed in HCT‐8 cells and in 2‐4‐6‐trinitrobenzen sulfonic acid induced colitis in rats mimicking inflammatory bowel disease by immunoprecipitation. No difference was observed in irritable bowel syndrome like model. In conclusion, we showed that a proteomic approach is suitable to identify ubiquitinated proteins and that UPS‐regulated expression of Grp75 and Grp78 may be involved in inflammatory response. Further studies should lead to the identification of ubiquitin ligases responsible for Grp75 and Grp78 ubiquitination.     

Identification of differentially expressed proteins by treatment with PUGNAc in 3T3‐L1 adipocytes through analysis of ATP‐binding proteome

O‐GlcNAc (2‐acetamino‐2‐deoxy‐β‐D‐glucopyranose), an important modification for cellular processes, is catalyzed by O‐GlcNAc transferase and O‐GlcNAcase. O‐(2‐acetamido‐2‐deoxy‐D‐glucopyranosylidene) amino‐N‐phenylcarbamate (PUGNAc) is a nonselective inhibitor of O‐GlcNAcase, which increases the level of protein O‐GlcNAcylation and is known to induce insulin‐resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNAc. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3T3‐L1 adipocytes and C2C12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP‐bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP‐binding protein families classified by PROSITE. The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3T3‐L1 adipocytes following treatment with PUGNAc. For label‐free quantitation, a gel‐assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data‐independent (671 proteins identified) and data‐dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide‐binding proteins and we focused on some nucleotide‐binding proteins, ubiquitin‐activation enzyme 1 (E1), Hsp70, vasolin‐containing protein (Vcp), and Hsp90, involved in ubiquitin‐proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNAc resulted in increased ubiquitination of proteins in a time‐dependent manner, and a decrease in both the amount of Akt and the level of phosphorylation of Akt, a key component in insulin signaling, through downregulation of Hsp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNAc. This result would provide insight into understanding functions of PUGNAc in 3T3‐L1 cells.     

The effect of simvastatin on the proteome of detergent‐resistant membrane domains: Decreases of specific proteins previously related to cytoskeleton regulation,calcium homeostasis and cell fate

Cell death induced by over‐activation of glutamate receptors occurs in different neuropathologies. Cholesterol depletors protect from neurotoxic over‐activation of glutamate receptors, and we have recently reported that this neuroprotection is associated with a reduction of the N‐methyl‐D ‐aspartate subtype of glutamate receptors in detergent‐resistant membrane domains (DRM). In the present study we used comparative proteomics to further identify which proteins, besides the N‐methyl‐D ‐aspartate receptor, change its percentage of association to DRM after treatment of neurons with simvastatin. We detected 338 spots in neuronal DRM subjected to 2‐DE; eleven of these spots changed its intensity after treatment with simvastatin. All 11 differential spots showed reduced intensity in simvastatin‐treated samples and were identified as adipocyte plasma membrane associated protein, enolase, calretinin, coronin 1a, f‐actin capping protein α1, f‐actin capping protein α2, heat shock cognate protein 71, malate dehydrogenase, n‐myc downregulated gene 1, prohibitin 2, Rab GDP dissociation inhibitor, translationally controlled tumor protein and voltage dependent anion selective channel protein 1. The proteins tested colocalized with the lipid raft marker caveolin‐1. Interestingly, the proteins we have identified in the present study had been previously reported to play a role in cell fate and, thus, they might represent novel targets for neuroprotection.     

KLF9 suppresses cell growth and induces apoptosis via the AR pathway in androgen-dependent prostate cancer cells

Kruppel-like factors (KLFs) play an important role in many biological processes including cell proliferation, differentiation and development. Our study showed that the level of KLF9 is lower in PCa cell lines compared to a benign prostate cell line; the androgen-independent cell line PC3 expresses significantly lower KLF9 than the androgen-dependent cell line, LNCaP. Forced overexpression of KLF9 suppressed cell growth, colony formation, and induced cell apoptosis in LNCaP cells. We also found that KLF9 expression was induced in response to apoptosis caused by flutamide, and further addition of dihydrotestosterone antagonized the action of flutamide and significantly decreased KLF9 expression. Furthermore, activation of the androgen receptor (AR) was inhibited by the overexpression of KLF9. Our research shows that KLF9 is lower in androgen-independent cell lines than in androgen-dependent cell lines; Overexpression of KLF9 dramatically suppresses the proliferation, anchorage-independent growth, and induces apoptosis in androgen-dependent cells; KLF9 inhibition on prostate cancer cell growth may be acting through the AR pathway. Our results therefore suggest that KLF9 may play a significant role in the transition from androgen-dependent to androgen-independent prostate cancer and is a potential target of prevention and therapy.     

Increases in cell size at START caused by hyperactivation of the cAMP pathway in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, passage through START, which commits cells to a new round of cell division, requires growth to a critical size. To examine the effect of hyperactivation of the cAMP pathway on cell size at START, a strain was constructed that is able to respond to exogenously added cAMP. In the presence of cAMP, this strain showed increased cell volume at bud emergence, suggesting that the critical cell size necessary for START is increased. In addition, a mutation that results in unregulated cAMP-dependent protein kinase (bcy1) caused increased cell size at START. These results indicate that hyperactivation of the cAMP pathway causes increases in cell size through cAMP-dependent protein kinase. Cells carrying a hyperactive allele of CLN3 (CLN3-2) also showed increased size at START in the presence of cAMP. These cells retained resistance to factor, however, suggesting that increases in cell size by cAMP are not due to a reduction of Cln3 activity. The observed increases in cell size due to hyperactivation of the cAMP pathway suggest that cell size modulation by nutrient conditions may be associated with a change of the activity of the cAMP pathway.     

Identification and characterization of highly expressed proteins in sperm cells of the marine mussel Mytilus edulis

Proteomic analysis on sperm has been restricted to only a few model organisms. We present here a 2DE PAGE proteome map of sperm cells from a nonmodel organism, the marine mussel Mytilus edulis, a free-spawning marine invertebrate with external fertilization. Ninety-six protein spots showing high expression were selected and of these 77 were successfully identified by nESI-MS analysis. Many of the identifications are relevant to sperm cell physiology and mtDNA functioning. The results and proteomics approach used are discussed in relation to their potential for advancing understanding of the unusual system of mtDNA inheritance described in Mytilus spp., and for the testing of evolutionary hypotheses pertaining to the role of fertilization in the speciation process.     

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis,motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA₁, LPA₂, and LPA₃ on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.     

Profiling of the human monocytic cell secretome by quantitative label-free mass spectrometry identifies stimulus-specific cytokines and proinflammatory proteins

The immune response to pathogens or injury relies on the concerted release of cytokines and proteins with biological activity important for host protection, host defense, and wound healing. Consequently, the secretome of immune cells provides a promising resource for discovery of specific molecular markers and targets for pharmacological intervention. Here, we employ label-free MS for unbiased, quantitative profiling of the human monocytic cell secretome under different proinflammatory stimuli. The quantitative secretome profiles reveal the highly stimulus-dependent cellular response and differential, specific secretion of more than 200 proteins, including important proinflammatory proteins and cytokines.     

Deltamethrin attenuates antioxidant defense system and induces the expression of heat shock protein 70 in rainbow trout

The current research aims to determine alterations in gene expression and enzymatic activity of fish antioxidant metabolism in response to pesticide administration. To this end, three different deltamethrin concentrations (0.25, 1, 2.5 μg/L) were administrated to rainbow trout (Oncorhynchus mykiss) at different time intervals (6, 12, 24, 48 and 72 h) in order to observe the influences of the pesticide on the activity of glutathione reductase, glucose 6-phosphate dehydrogenase, 6-ghosphogluconate dehydrogenase, and the expression of Hsp70 gene. We observed that the activities of the enzymes decreased with increasing deltamethrin concentrations and exposure time. The pesticide had more inhibitory effects on gill enzymes than those of muscle, liver and kidney. In addition, we detected that deltamethrin increased the expression of the stress-related protein Hsp70 with significant fold-chance values. The efficiency rate was 96.4% which is equal to 1.96 calculated via conversion formula used to calculate fold-chance value. We conclude that deltamethrin causes oxidative stress in fish both at protein and mRNA levels.     

Phosphorylation of the SSBP2 and ABL proteins by the ZNF198‐FGFR1 fusion kinase seen in atypical myeloproliferative disorders as revealed by phosphopeptide‐specific MS

The ZNF198‐fibroblast growth factor receptor‐1 (FGFR1) fusion kinase is a constitutively activated tyrosine kinase associated with a specific atypical myeloproliferative disease. The chimeric protein localizes to the cytoplasm, unlike the wild type FGFR1 receptor kinase, and presumably inappropriately phosphorylates specific targets as part of the oncogenic signaling cascade. Other than known targets of the FGFR1 kinase itself, few specific targets of ZNF198‐FGFR1 have been identified. Using a genetically engineered HEK 293 cell system, we have identified proteins that are specifically phosphorylated in the presence of the fusion kinase using anti‐phosphotyrosine immunoprecipitation and MS. Compared with 293 cells expressing exongenous wild type FGFR1, ZNF198‐FGFR1 is associated with phosphorylation of several proteins including SSBP2, ABL, FLJ14235, CALM and TRIM4 proteins. The specificity of the phosphorylation events in the SSBP2 and ABL proteins, which have previously been implicated in leukemogenesis, was further confirmed independently using immunoprecipitation with protein‐specific antibodies and Western blotting. The MS analysis also identified the phosphorylation events in the ZNF198 moiety in the chimeric protein that might be related to its function. These studies identify the intersection of several different leukemia‐related pathways in the development of this myeloproliferative disorder and provide new insights into the substrates of FGFR1 under defined conditions.     

Coupling proteomics and transcriptomics in the quest of subtype‐specific proteins in breast cancer

Breast‐cancer subtypes present with distinct clinical characteristics. Therefore, characterization of subtype‐specific proteins may augment the development of targeted therapies and prognostic biomarkers. To address this issue, MS‐based secretome analysis of eight breast cancer cell lines, corresponding to the three main breast cancer subtypes was performed. More than 5200 non‐redundant proteins were identified with 23, four, and four proteins identified uniquely in basal, HER2‐neu‐amplified, and luminal breast cancer cells, respectively. An in silico mRNA analysis using publicly available breast cancer tissue microarray data was carried out as a preliminary verification step. In particular, the expression profiles of 15 out of 28 proteins included in the microarray (from a total of 31 in our subtype‐specific signature) showed significant correlation with estrogen receptor (ER) expression. A MS‐based analysis of breast cancer tissues was undertaken to verify the results at the proteome level. Eighteen out of 31 proteins were quantified in the proteomes of ER‐positive and ER‐negative breast cancer tissues. Survival analysis using microarray data was performed to examine the prognostic potential of these selected candidates. Three proteins correlated with ER status at both mRNA and protein levels: ABAT, PDZK1, and PTX3, with the former showing significant prognostic potential.     

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti‐phosphotyrosine and anti‐phosphoserine antibodies following 2‐DE in conjunction with double channel laser‐induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans‐membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.     

The Shigella dysenteriae serotype 1 proteome,profiled in the host intestinal environment,reveals major metabolic modifications and increased expression of invasive proteins

Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. We present the first comprehensive proteome analysis of this pathogen, profiling proteins from bacteria cultured in vitro and bacterial isolates from the large bowel of infected gnotobiotic piglets (in vivo). Overall, 1061 distinct gene products were identified. Differential display analysis revealed that SD1 cells switched to an anaerobic energy metabolism in vivo. High in vivo abundances of amino acid decarboxylases (GadB and AdiA) which enhance pH homeostasis in the cytoplasm and protein disaggregation chaperones (HdeA, HdeB and ClpB) were indicative of a coordinated bacterial survival response to acid stress. Several type III secretion system effectors were increased in abundance in vivo, including OspF, IpaC and IpaD. These proteins are implicated in invasion of colonocytes and subversion of the host immune response in S. flexneri. These observations likely reflect an adaptive response of SD1 to the hostile host environment. Seven proteins, among them the type III secretion system effectors OspC2 and IpaB, were detected as antigens in Western blots using piglet antisera. The outer membrane protein OmpA, the heat shock protein HtpG and OspC2 represent novel SD1 subunit vaccine candidates and drug targets.     

Comparative proteomics analysis reveals roles for FADD in the regulation of energy metabolism and proteolysis pathway in mouse embryonic fibroblast

Fas‐associated death domain‐containing protein (FADD) is a classical apoptotic pathway adaptor. Further studies revealed that it also plays essential roles in nonapoptotic processes, which is assumed to be regulated by its phosphorylation. However, the exact mechanisms are still poorly understood. To study the nonapoptotic effects of FADD, a comprehensive strategy of proteomics identification combined with bioinformatic analysis was undertaken to identify proteins differentially expressed in three cell lines containing FADD and its mutant, FADD‐A and FADD‐D. The cell lines were thought to bear wild‐type FADD, unphosphorylated FADD mimic and constitutive phosphorylated FADD mimic, respectively. A total of 47 proteins were identified to be significantly changed due to FADD phosphorylation. Network analysis using MetaCore^TM identified a number of changed proteins that were involved in cellular metabolic process, including lipid metabolism, fatty acid metabolism, glycolysis, and oxidative phosphorylation. The finding that FADD‐D cell line showed an increase in fatty acid oxidation argues that it could contribute to the leaner phenotype of FADD‐D mice as reported previously. In addition, six proteins related to the ubiquitin‐proteasome pathway were also specifically overexpressed in FADD‐D cell line. Finally, the c‐Myc gene represents a convergent hub lying at the center of dysregulated pathways, and was upregulated in FADD‐D cells. Taken together, these studies allowed us to conclude that impaired mitochondrial function and proteolysis might play pivotal roles in the dysfunction associated with FADD phosphorylation‐induced disorders.