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PubMedDate not supplied

Proteomic analysis of pancreatic ductal carcinoma cells after combined treatment with gemcitabine and trichostatin A.

The human pancreatic adenocarcinoma cell line T3M4 has been treated with two agents, gemcitabine (2',2'-difluorodeoxycytidine, a drug interfering with DNA synthesis) and trichostatin A (a drug interfering with histone acetylation), both separately and in association. The association of the two drugs showed a marked cooperative effect in inhibiting proliferation and inducing apoptosis of the cells. In an effort to identify differentially expressed proteins in the different drug treatments, the proteomic expression has been studied by two-dimensional gel electrophoresis comparing untreated cells with cells treated with trichostatin A and/or gemcitabine. A total of 81 differentially expressed polypeptide chains have been visualized by setting a 2.5-fold threshold value. Of these, 56 were identified via MALDI-TOF and Q-TOF MS analyses. Most of the regulated proteins are involved in two major biological processes, namely apoptotic cell death and proliferation. Our results demonstrate that the level of activation/repression of the proteins involved in these processes correlates with the growth inhibition and the apoptotic response of the cells subjected to single or combined drug treatment.

Antibiotics, Antineoplastic↗

PubMed2004

In vitro and in silico processes to identify differentially expressed proteins.

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.

Body Fluids↗

PubMed2005

Protein nitration in a mouse model of familial amyotrophic lateral sclerosis: possible multifunctional role in the pathogenesis.

Multiple mechanisms have been proposed to contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, including oxidative stress. Early evidence of a role for oxidative damage was based on the finding, in patients and murine models, of high levels of markers, such as free nitrotyrosine (NT). However, no comprehensive study on the protein targets of nitration in ALS has been reported. We found an increased level of NT immunoreactivity in spinal cord protein extracts of a transgenic mouse model of familial ALS (FALS) at a presymptomatic stage of the disease compared with age-matched controls. NT immunoreactivity is increased in the soluble fraction of spinal cord homogenates and is found as a punctate staining in motor neuron perikarya of presymptomatic FALS mice. Using a proteome-based strategy, we identified proteins nitrated in vivo, under physiological or pathological conditions, and compared their level of specific nitration. alpha- and gamma-enolase, ATP synthase beta chain, and heat shock cognate 71-kDa protein and actin were overnitrated in presymptomatic FALS mice. We identified by matrix-assisted laser desorption/ionization mass spectrometry 16 sites of nitration in proteins oxidized in vivo. In particular, alpha-enolase nitration at Tyr(43), target also of phosphorylation, brings additional evidence on the possible interference of nitration with phosphorylation. In conclusion, we propose that protein nitration may have a role in ALS pathogenesis, acting directly by inhibiting the function of specific proteins and indirectly interfering with protein degradation pathways and phosphorylation cascades.

Amino Acid Substitution↗

PubMed2006

A proteomic approach to analysing responses of Arabidopsis thaliana callus cells to clinostat rotation.

Callus cells of Arabidopsis thaliana (cv. Landsberg erecta) were exposed for 8 h to a horizontal clinostat rotation (H, simulated weightlessness), a vertical clinostat rotation (V, clinostat control), or a stationary control (S) growth condition. The amount of glucose and fructose apparently decreased, while starch content increased in the H compared with the V- and S-treated cells. In order to investigate the influences of clinostat rotation on the cellular proteome further, the proteome alterations induced by horizontal and vertical clinostat rotation have been comparatively analysed by high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 80 protein spots showed quantitative and qualitative variations that were significantly (P <0.01) and reproducibly different between the clinorotated (H or V) and the stationary control samples. Protein spots excised from 2-D gels were analysed by microbe high performance liquid chromatography-ion trap-mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. 18 protein spots, which showed significant expression alteration only under the H condition compared with those under V and S conditions, were identified. Of these proteins, seven were involved in stress responses, and four protein spots were identified as key enzymes in carbohydrate metabolism and lipid biosynthesis. Two reversibly glycosylated cell wall proteins were down-regulated in the H samples. Other proteins such as protein disulphide isomerase, transcription initiation factor IIF, and two ribosomal proteins also exhibited altered expression under the H condition. The data presented in this study illustrate that clinostat rotation of Arabidopsis callus cells has a significant impact on the expression of proteins involved in general stress responses, metabolic pathways, gene activation/transcription, protein synthesis, and cell wall biosynthesis.

Arabidopsis↗

PubMed2005

Proteomic analysis of differential protein expression in human atherosclerotic plaque progression.

In this study, differential protein expression was assessed during human atherosclerotic plaque progression. A multifaceted approach was used in which differential protein expression was studied by two-dimensional (2D) gel electrophoresis and validated in individual patients using western blotting and immunohistochemistry. 2D profiles of whole-mount advanced stable lesions were compared to those of plaques containing a thrombus. Mass spectrometry analysis identified vinexin-beta and alpha1-antitrypsin (AAT) in the same spot that was differentially expressed in plaques with a thrombus. Immunohistochemistry and western blotting showed limited expression of both vinexin-beta and AAT in early lesions, whereas high expression of both proteins was found in advanced lesions. Differential expression of vinexin-beta in lesions with a thrombus compared to stable plaques could not be confirmed, indicating the importance of validation of proteomic analysis. For AAT, western blotting of 2D gels revealed expression of six isoforms in advanced plaques, one of which was confirmed to be solely expressed in thrombus-containing plaques. In conclusion, vinexin-beta is expressed in advanced human atherosclerotic plaques, but differential expression of this protein in lesions with a thrombus versus stable plaques could not be confirmed. However, this analysis revealed expression of six isoforms of AAT in advanced plaques, one of which was uniquely expressed in thrombus-containing plaques.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Proteins associated with Cisplatin resistance in ovarian cancer cells identified by quantitative proteomic technology and integrated with mRNA expression levels.

Nearly all women diagnosed with ovarian cancer receive combination chemotherapy including cis- or carboplatin. Despite high initial response rates, resistance to cisplatin develops in roughly one-third of women during primary treatment and in all women treated for recurrent disease. ICAT coupled with tandem MS is a quantitative proteomic technique for high throughput protein expression profiling of complex protein mixtures. Using ICAT/MS/MS we profiled the nuclear, cytosolic, and microsomal fractions obtained from IGROV-1 [corrected] (cisplatin-sensitive) and IGROV-1/CP [corrected] (cisplatin-resistant) ovarian cancer cell lines. The proteomes of cisplatin-sensitive and -resistant ovarian cancer cells were compared, and protein expression was correlated with mRNA expression profiles. A total of 1117 proteins were identified and quantified. The relative expression of 121 of these varied between the two cell lines. Sixty-three proteins were overexpressed in cisplatin-sensitive, and 58 were over expressed in cisplatin-resistant cells. Examples of proteins at least 5-fold overexpressed in resistant cells and with biological relevance to cancer include cell recognition molecule CASPR3 (13.3-fold), S100 protein family members (8.7-fold), junction adhesion molecule Claudin 4 (7.2-fold), and CDC42-binding protein kinase beta (5.4-fold). Examples of cancer-related proteins at least 5-fold overexpressed in sensitive cells include hepatocyte growth factor inhibitor 1B (13.3-fold) and programmed cell death 6-interacting protein (12.7-fold). The direction of changes in expression levels between proteins and mRNAs were not always in the same direction, possibly reflecting posttranscriptional control of protein expression. We identified proteins whose expression profiles correlate with cisplatin resistance in ovarian cancer cells. Several proteins may be involved in modulating response to cisplatin and have potential as markers of treatment response or treatment targets.

Cell Line, Tumor↗

PubMed2004

Microarray platform for profiling enzyme activities in complex proteomes.

Activity-based protein profiling (ABPP) is a chemical method that utilizes active-site-directed probes to determine the functional state of enzymes in complex proteomes. Probe-labeled enzymes are typically detected by in-gel fluorescence scanning, a robust technique that nonetheless exhibits some key deficiencies, including limited sensitivity and resolution, as well as ambiguity regarding the molecular identity of the enzymes under investigation. Here, we report a microarray platform for ABPP that addresses these limitations. In this platform, proteomes are treated with ABPP probes in solution, after which labeled enzymes are captured and visualized on glass slides displaying an array of anti-enzyme antibodies. We show that ABPP microarrays exhibit superior sensitivity and resolution compared to gel-based methods, permitting the parallel analysis of several enzyme activities in proteomes, including cancer-associated proteases such as urokinase, matrix metalloproteinase-9, and prostate-specific antigen.

Antibodies↗

PubMed2007

Spatial learning induces predominant downregulation of cytosolic proteins in the rat hippocampus.

Spatial learning is known to depend on protein synthesis in the hippocampus. Whereas the role of the hippocampus in spatial memory is established, the biochemical and molecular mechanisms underlying this process are poorly understood. To comprehend the complex pattern of protein expression induced by spatial learning, we analyzed alterations in the rat hippocampus proteome after 7 days of spatial learning in the Morris water maze. Forty Wistar rats were randomized into two groups. Animals of group A learned to localize a hidden platform in the water maze. Animals of group B served as controls and spent exactly the same time in the water maze as animals of group A. However, no platform was used in this test and the rats could not learn to localize the target. After the last trial, hydrophilic proteins from the hippocampus were isolated. A proteome-wide study was performed, based on two-dimensional gel electrophoresis and mass spectrometry. Compared with non-learning animals, 53 (70%) proteins were downregulated and 23 (30%) proteins were upregulated after 7 days in rats with spatial learning. The overall changes in protein expression, as quantified by the induction factor, ranged from -1.62 (downregulation to 62%) to 2.10 (upregulation by 110%) compared with controls (100%). Most identified proteins exhibit known functions in vesicle transport, cytoskeletal architecture, and metabolism as well as neurogenesis. These findings indicate that learning in the Morris water maze has a morphological correlate on the proteome level in the hippocampus.

Animals↗

PubMed2026

Proteomic analysis of ammonia-induced stress in Chinese hamster ovary (CHO) cell cultures.

Ammonia (NH₃) and its ionic form ammonium (NH₄+) are both metabolic waste products and essential nitrogen sources within Chinese hamster ovary (CHO) cell cultures. Although necessary for amino acid synthesis, excessive accumulation in the extracellular environment can exert stress, reducing cell proliferation and impairing the efficiency of recombinant protein production. Proper endoplasmic reticulum (ER) function is critical for CHO cells as biotherapeutic producers. Previous work has linked elevated ammonia concentrations to reduced productivity via altered N-glycosylation pathways, but its broader effects on ER biology remain unclear. In this study, we applied high-resolution mass spectrometry to perform a comprehensive analysis of changes in the ER proteome in CHO cells exposed to two ammonia concentrations, 10 mM and 30 mM, 48 and 120 h after supplementation. Both conditions suppressed cell growth and reduced product titre; however, the 10 mM supplementation resulted in a minor increase in specific cell productivity. Gene Ontology analysis revealed that ammonia strongly affected the tricarboxylic acid cycle, as well as key metabolic, catabolic and biogenetic processes. Several ER membrane proteins, including HMGCR and PREB, were consistently downregulated. In extended cultures, transmembrane proteins linked to Golgi-transport were upregulated, while vesicle transport associated proteins were downregulated, indicating altered intracellular trafficking. SIGNIFICANCE: This study provides a novel perspective on CHO cell biology under environmental stress by investigating the impact of ammonia accumulation in culture. Despite its presence in CHO culture, ammonia has been relatively under-investigated, compared to other culture conditions. Using high-throughput mass spectrometry for comprehensive proteomic profiling, we characterise the cellular response to ammonia build-up with a level of depth not previously applied to the study of this biological stressor. By specifically analysing proteins localised to the ER, we identify candidate pathways and molecular mechanisms that contribute to reduced CHO cell growth and productivity, offering insights directly relevant to industrial bioprocessing conditions. The link between ammonia concentration and a decrease in productivity has previously been linked to genes involved in N-glycosylation of the recombinant biotherapeutic, but the full extent of ammonia stress on ER function has not yet been investigated. These methods were applied to two IgG producing CHO cell lines to allow for comparison of cell line specific stress adaptations, as well as comparing the short- and long-term effects of excess ammonia.

Proteomics↗

PubMedDate not supplied

Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS.

This study uses multiple enzyme digests to increase the sequence coverage of proteins identified by the shotgun sequencing approach to proteomic analysis. The enzymes used were trypsin, Lys-C, and Asp-N, which cleave at arginine and lysine residues, lysine, and aspartic acid residues, respectively. This approach was evaluated with the glycoprotein, tissue plasminogen activator, t-PA and gave enhanced sequence coverage, compared with a single enzymatic digest. The approach was then evaluated with a complex proteomic sample, namely plasma. It was found that trypsin and Lys-C were able to detect overlapping but distinct sets of proteins and a digital recombination of the data gave a significant increase in both the number of protein identifications as well as an increase in the number of peptides identified per protein (which improves the certainty of the assignment).

Amino Acid Sequence↗

PubMed2026

Harnessing metabolomics and proteomics in a clinical trial for pulmonary arterial hypertension: insights from post-hoc analysis of the REHAB-PH trial.

BACKGROUND: The significant clinical and molecular heterogeneity of pulmonary arterial hypertension (PAH) poses challenges in identifying effective therapies. Advanced multidimensional profiling offers an opportunity to capture molecular responses and assess biomarker stability, yet its application in randomised trials remains limited. METHODS: We evaluated the multi-omic profiles of participants with PAH in a randomised, placebo-controlled trial of famotidine. Plasma metabolomic and proteomic profiling was performed at enrolment and 24 weeks. Baseline profiles were compared between treatment arms to assess randomisation balance. Intraclass correlation coefficients quantified within-subject stability over time. Linear regression models adjusting for age, sex, body mass index and PAH aetiology evaluated famotidine's molecular effects. False discovery rate was controlled for multiple comparisons. FINDINGS: For the 79 participants, baseline multi-omic profiles were similar between groups. At 24 weeks, 34 and 37 participants remained in the famotidine and placebo groups respectively. The placebo group showed high molecular stability, while greater variability was observed in the famotidine group. Famotidine treatment was associated with significant changes across 191 proteomic pathways (q-value <0.05), but no metabolomic changes remained significant after multiple-testing correction. INTERPRETATION: Integrating multi-omics into a prospective clinical trial is feasible and yields stable longitudinal profiles in the absence of intervention. While famotidine did not yield clinical benefit, associated proteomic changes illustrate how molecular profiling can reveal treatment-related biology and inform future trial design. These findings highlight the broader utility of multi-omics for evaluating drug responses and identifying molecular endotypes in PAH and beyond. FUNDING: US National Institutes of Health.

Humans↗

PubMed2004

Proteomic identification of brainstem cytosolic proteins in a neuropathic pain model.

Neuropathic pain involves co-regulation of many genes and their translational products in both peripheral and central nervous system. We used proteomics approaches to investigate expressional changes in cytosolic protein levels in rat brainstem tissues following ligation of lumbar 5 and 6 (L5, L6) spinal nerves, which generates a model of peripheral neuropathic pain (NP). Proteins from brainstem tissue homogenates of NP and SHAM animals were fractionated by two-dimensional (2-DE) gel electrophoresis to produce a high-resolution map of the brainstem soluble proteins. Proteins showing altered expression levels between NP and SHAM were selected. Isolated proteins were in-gel trypsin-digested and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Using the mass spectrometric data, we were able to identify 17 proteins of interest through searches of the Swiss-Prot and NCBi nonredundant protein sequence database. Several of the identified proteins, including fatty acid binding protein-brain (FABP-B), major histocompatibility complex (MHC) class 1, T-cell receptor (TCR) alpha chain, and interleukin-1 (IL-1), showed significantly higher levels in the NP rat brainstem. Proteomic analysis has identified several proteins with differential expression levels in NP as compared to SHAM. However, the function of the proteins identified is postulated; therefore, further experiments are required to determine the true role of each protein in NP.

Animals↗

PubMed2006

[Comparison between protein detection methods for two-dimensional gel electrophoresis].

The aim of this study was to compare and analyze protein staining in order to select the optimal staining method for proteomic research. Proteins from acute promyelocytic leukemia cell line NB4 and protein molecular weight marker were separated respectively by 2-D or 1-D electrophoresis and detected respectively by the typical Coomassie brilliant blue, the colloidal Coomassie brilliant blue, the modified Coomassie brilliant blue and the silver staining protocols. The protein detection sensitivity, compatibility with mass spectrometry (MS) and facility of the four staining protocols were compared. The results indicated that the silver staining exhibited the highest sensitivity and MS showed the lowest compatibility 10% of protein identification rate. The detection sensitivity of the modified Coomassie brilliant blue staining was superior to that of other two Coomassie brilliant blue stainings, close to but lower than the silver staining, however the compatibility with MS was better (protein identification rate about 55%). It is concluded that the protein detection sensitivity of the modified Coomassie brilliant blue staining is high, and its compatibility with MS is better, this modified Coomassie brilliant blue staining is an optimal staining method for proteomic research.

Coloring Agents↗

PubMed2006

Proteomic analysis of enamel matrix using a two-dimensional protein fractionation system.

Our objectives in this study were to perform separate proteomic analyses of porcine soft and hard enamel matrices, using the ProteomeLab PF-2D System, to compare the contents of the hard and soft enamel and to identify matrix constituents that are absent from the early maturation stage. Developing first permanent molars were dissected from 6-month-old pigs. Both immature and mature enamel samples were obtained by scraping the secretory-stage (soft) and maturation-stage (hard) enamel, respectively. Enamel matrix samples were sequentially extracted and fractionated with 50 mM phosphate buffer (pH 7.4) and then with 50 mM carbonate buffer (pH 10.8). The neutral enamel extract was separated into four fractions by successive ammonium sulfate precipitations. The alkaline enamel extract was separated into four fractions by ion-exchange chromatography. These eight extracts from both the soft and hard enamel were injected for chromatofocusing. Soft enamel fractions containing constituents absent from the hard enamel were further separated by reverse-phase high-performance liquid chromatography. The major soft enamel constituents absent from the hard enamel were acidic glycoproteins, corresponding to the 32-kDa enamelin, and the 29-, 27-, 15-, 13-, 8- and 6-kDa C-terminal fragments of ameloblastin. Loss of these glycoproteins is associated with a post-transition increase in enamel mineralization.

Amelogenesis↗

PubMed2003

Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis.

Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic in-solution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200 nL/min. Mass measurement accuracies smaller than 3 ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis.

Animals↗

PubMed2006

Trade-off between high sensitivity and increased potential for false positive peptide sequence matches using a two-dimensional linear ion trap for tandem mass spectrometry-based proteomics.

Two-dimensional linear ion trap mass spectrometers are rapidly becoming the new workhorse instruments for shotgun proteomic analysis of complex peptide mixtures. The objective of this study was to compare the potential for false positive peptide sequence matches between a two-dimensional ion trap instrument and a traditional, three-dimensional ion trap instrument. Through the comparative analysis of a complex protein sample, we found that in order to minimize false positive sequence matches, sequence match scoring criteria must be more stringent for data from the two-dimensional ion trap compared to the three-dimensional ion trap data. Given this increased potential for false positives, we also investigated two potential filtering strategies to reduce the false positive matches for data derived from the two-dimensional ion trap, including trypsin enzyme cleavage filtering, and the addition of peptide physicochemical information as a constraint, specifically peptide isoelectric point. The results described here provide a cautionary tale to researchers, demonstrating the need for careful analysis of MS/MS data from this new class of ion trap instruments, as well as the effectiveness of trypsin enzyme cleavage filtering and peptide pI information in maximizing high confidence protein identifications from this powerful proteomic instrumentation.

Amino Acid Sequence↗

PubMedDate not supplied

DBParser: web-based software for shotgun proteomic data analyses.

We describe a web-based program called 'DBParser' for rapidly culling, merging, and comparing sequence search engine results from multiple LC-MS/MS peptide analyses. DBParser employs the principle of parsimony to consolidate redundant protein assignments and derive the most concise set of proteins consistent with all of the assigned peptide sequences observed in an experiment or series of experiments. The resulting reports summarize peptide and protein identifications from multidimensional experiments that may contain a single data set or combine data from a group of data sets, all related to a single analytical sample. Additionally, the results of multiple experiments, each of which may contain several data sets, can be compared in reports that identify features that are common or different. DBParser actively links to the primary mass spectral data and to public online databases such as NCBI, GO, and Swiss-Prot in order to structure contextually specific reports for biologists and biochemists.

Amino Acid Sequence↗

PubMed2002

Establishing two-dimensional gels for the analysis of Leishmania proteomes.

Several different sample preparation methods for two-dimensional electrophoresis (2-DE) analysis of Leishmania parasites were compared. From this work, we were able to identify a solubilization method using Nonidet P-40 as detergent, which was simple to follow, and which produced 2-DE gels of high resolution and reproducibility.

Animals↗