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PubMed2005

Development and application of proteomics technologies in Saccharomyces cerevisiae.

Proteomics research focuses on the identification and quantification of "all" proteins present in cells, organisms or tissue. Proteomics is technically complicated because it encompasses the characterization and functional analysis of all proteins that are expressed by a genome. Moreover, because the expression levels of proteins strongly depend on complex regulatory systems, the proteome is highly dynamic. This review focuses on the two major proteomics methodologies, one based on 2D gel electrophoresis and the other based on liquid chromatography coupled to mass spectrometry. The recent developments of these methodologies and their application to quantitative proteomics are described. The model system Saccharomyces cerevisiae is considered to be the optimal vehicle for proteomics and we review studies investigating yeast adaptation to changes in (nutritional) environment.

Chromatography, Liquid↗
PubMed2025

Proteomic insights into platelet dysregulation and pathogenic mechanisms of chronic thromboembolic pulmonary hypertension.

BACKGROUND: Undissolved thrombus blocks the pulmonary arteries in chronic thromboembolic pulmonary hypertension (CTEPH), a potentially fatal illness that raises pulmonary resistance, causes right heart failure, and even results in death. Although platelets are linked to vascular dysfunction and thrombus formation, it is yet unknown what precise proteome alterations and mechanistic roles they play in CTEPH. METHODS: We extracted platelet-rich plasma from peripheral blood and separated the plasma to obtain enriched platelet pellet (EPP). Quantitative proteomics was used to examine EPP from CTEPH patients and healthy controls using mass spectrometry. The relationship between protein levels and clinical markers of right heart function was examined. Platelet activity, morphology, and interactions with other blood components were evaluated using transmission electron microscopy, immunofluorescence, and flow cytometry. RESULTS: The proteomic investigation found that 179 proteins were differentially expressed in CTEPH patients. The analysis revealed that these proteins were involved in crucial processes such as complement and coagulation cascades, phagosome, and neutrophil extracellular trap (NET) formation. Elevated proteins, specifically NOX2, PAD4, ITGB2, and HMGB1, have been associated to platelet-neutrophil aggregates and NET formation. In addition, enhanced P-selectin expression in platelets and plasma confirmed greater platelet activation in CTEPH patients. Notably, PAD4 and NOX2 levels showed a substantial correlation with hemodynamic parameters and right heart dysfunction. MPO-DNA, a NET marker associated with P-selectin and ITGB2 expression, was discovered in higher concentrations in CTEPH patients' plasmas. CONCLUSION: Platelet aggregation and activation in CTEPH encourage the formation of NETs, which advances the disease and prolongs thrombus. Right heart insufficiency and hemodynamic markers had a strong correlation with PAD4 and NOX2 levels, indicating that these biomarkers may be employed to assess the severity and prognosis of CTEPH disease and offer a fresh approach to targeted treatment. The results highlight the need for additional study to elucidate platelet-mediated pathways and create therapies for CTEPH that target platelets.

Humans↗
PubMed2006

Phosphoproteomic analysis of Her2/neu signaling and inhibition.

Her2/neu (Her2) is a tyrosine kinase belonging to the EGF receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. We sought to characterize Her2 signal transduction pathways further by using MS-based quantitative proteomics. Stably transfected cell lines overexpressing Her2 or empty vector were generated, and the effect of an EGFR and Her2 selective tyrosine kinase inhibitor, PD168393, on these cells was characterized. Quantitative measurements were obtained on 462 proteins by using the SILAC (stable isotope labeling with amino acids in cell culture) method to monitor three conditions simultaneously. Of these proteins, 198 showed a significant increase in tyrosine phosphorylation in Her2-overexpressing cells, and 81 showed a significant decrease in phosphorylation. Treatment of Her2-overexpressing cells with PD168393 showed rapid reversibility of the majority of the Her2-triggered phosphorylation events. Phosphoproteins that were identified included many known Her2 signaling molecules as well as known EGFR signaling proteins that had not been previously linked to Her2, such as Stat1, Dok1, and delta-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site in Her2, Y877, which is located in the activation loop of the kinase domain, is distinct from the known C-terminal tail autophosphorylation sites, and may have important implications for regulation of Her2 signaling. Network modeling, which combined phosphoproteomic results with literature-curated protein-protein interaction data, was used to suggest roles for some of the previously unidentified Her2 signaling proteins.

Amino Acid Sequence↗
PubMed2005

Computational analysis of shotgun proteomics data.

Proteomics technology is progressing at an incredible rate. The latest generation of tandem mass spectrometers can now acquire tens of thousands of fragmentation spectra in a matter of hours. Furthermore, quantitative proteomics methods have been developed that incorporate a stable isotope-labeled internal standard for every peptide within a complex protein mixture for the measurement of relative protein abundances. These developments have opened the doors for 'shotgun' proteomics, yet have also placed a burden on the computational approaches that manage the data. With each new method that is developed, the quantity of data that can be derived from a single experiment increases. To deal with this increase, new computational approaches are being developed to manage the data and assess false positives. This review discusses current approaches for analyzing proteomics data by mass spectrometry and identifies present computational limitations and bottlenecks.

Algorithms↗
PubMed2025

Multi-omic integration with human dorsal root ganglia proteomics highlights TNFα signalling as a relevant sexually dimorphic pathway.

The peripheral nervous system (PNS) plays a critical role in pathological conditions, including chronic pain disorders, that manifest differently in men and women. To investigate this sexual dimorphism at the molecular level, we integrated quantitative proteomic profiling of human dorsal root ganglia (hDRG) and peripheral nerve tissue into the expanding omics framework of the PNS. Using data-independent acquisition (DIA) mass spectrometry, we characterized a comprehensive proteomic profile, validating tissue-specific differences between the hDRG and peripheral nerve. Through multi-omic analyses and in vitro functional assays, we identified sex-specific molecular differences, with TNFα signalling emerging as a key sexually dimorphic pathway with higher prominence in men. Genetic evidence from genome-wide association studies further supports the functional relevance of TNFα signalling in the periphery, while clinical trial data and meta-analyses indicate a sex-dependent response to TNFα inhibitors. Collectively, these findings underscore a functionally sexual dimorphism in the PNS, with direct implications for sensory and pain-related clinical translation.

Humans↗
PubMed2006

Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells.

Study of stem cells may reveal promising treatment for diseases. The fate and function of transplanted stem cells remain poorly defined. Recent studies demonstrate that reporter genes can monitor real-time survival of transplanted stem cells in living subjects. We examined the effects of a novel and versatile triple fusion (TF) reporter gene construction on embryonic stem (ES) cell function by proteomic analysis. Murine ES cells were stably transduced with a self-inactivating lentiviral vector containing fluorescence (firefly luciferase; Fluc), bioluminescence (monomeric red fluorescence protein; mRFP), and positron emission tomography (herpes simplex virus type 1 truncated thymidine kinase; tTK) reporter genes. Fluorescence-activated cell sorting (FACS) analysis isolated stably transduced populations. TF reporter gene effects on cellular function were evaluated by quantitative proteomic profiling of control ES cells versus ES cells stably expressing the TF construct (ES-TF). Overall, no significant changes in protein quantity were observed. TF reporter gene expression had no effect on ES cell viability, proliferation, and differentiation capability. Molecular imaging studies tracked ES-TF cell survival and proliferation in living animals. In summary, this is the first proteomic study, demonstrating the unique potential of reporter gene imaging for tracking ES cell transplantation non-invasively, repetitively, and quantitatively.

Animals↗
PubMed2006

The application of DIGE-based proteomics to renal physiology.

Proteomics is seeing increasing use as a means of identifying new mechanistic hypotheses in physiology. Proteomics based on two-dimensional electrophoresis (2-DE) has recently been optimized with the development of Difference Gel Electrophoresis (DIGE). In DIGE-based proteomics, the experimental and control samples are derivatized with different fluorophores and are run in the same gel, thereby minimizing technical variation. DIGE is currently one of the few techniques to perform quantitative proteomics, generating a statistical output to differences in protein abundances. In this review, we discuss the principles of DIGE-based proteomics, including sample preparation, 2-DE, statistical analysis of 2D-gels, and mass spectrometry. Strengths and weaknesses of DIGE are discussed, including possible solutions to overcome certain limitations, such as the identification of low abundance and integral membrane proteins. In addition, we provide a brief synopsis of our recent experiments in which DIGE-based proteomics was applied to study vasopressin signaling in the renal collecting duct. Finally, we illustrate how quantification based on the DIGE approach combined with bioinformatics may facilitate the study of systems biology of the kidney.

Animals↗
PubMed2005

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis.

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Animals↗
PubMed2026

Early proteomic and metabolic signatures of liver and eye in OAT-deficient mice.

Ornithine aminotransferase (OAT) deficiency causes hyperornithinemia and gyrate atrophy (GA) of the choroid and retina, a rare inherited retinal degeneration. To understand the early molecular changes that make the eye susceptible to damage, we performed quantitative proteomic and metabolomic profiling of liver, retina, and retinal pigment epithelium and choroid (RPE/Cho) from OAT-deficient (Oatrhg) mice prior to detectable vision impairment. In addition to reduced OAT expression and elevated ornithine, methylation-related metabolites such as N(6)-methyl-lysine were altered in all examined tissues of Oatrhg mice. In the liver, excess ornithine was directed into urea cycle metabolism, together with altered expression of detoxification enzymes and histone H2B proteins. In contrast, the retina showed minimal proteomic changes but pronounced alterations in amino acid pathways that support glutamate homeostasis. The RPE/Cho demonstrated the most extensive proteomic changes, particularly in mitochondrial metabolism, cytoskeleton, and extracellular matrix, along with changes in metabolites involved in lysine metabolism, energy metabolism, and antioxidant capacity. Incubation with 13C lysine demonstrated that lysine was primarily degraded in RPE/Cho but not the retina, and ornithine enhanced lysine degradation in an OAT-dependent manner. Together, these findings highlight common and tissue-specific impacts of OAT on the liver and ocular tissues and provide insight into early molecular changes that contribute to the selective vulnerability of the eye in GA. Proteomics data are available via ProteomeXchange (PXD063614) and metabolomics data via MassIVE repository (MSV000101103).

Animals↗
PubMedDate not supplied

Comparative proteomics of glycoproteins based on lectin selection and isotope coding.

Lectins have been widely used in glycan structure analysis. The studies described here exploit this fact to select glycopeptides carrying disease-associated modifications in their oligosaccharides. Coupling lectin affinity selection with recent advances in stable isotope coding for quantitative proteomics allowed a comparative proteomics method to be developed for examining aberrant glycosylation in cancer. Control and experimental samples were individually tryptic digested and differentially coded with stable isotope coding agents before they were mixed and affinity selected with a lectin affinity chromatography column. Glycopeptides carrying an alpha-L-fucose residue were selected with Lotus tetragonolobus agglutinin (LTA) immobilized on a chromatography matrix. Because the oligosaccharides of glycoproteins are generally heterogeneous and often of unknown structure, it was necessary to deglycosylate the selected peptides with PNGase F before they could be compared to sequences in DNA and protein databases. After deglycosylated peptides were transferred to a reversed phase chromatography (RPC) column and fractionated by gradient elution with increasing amounts of acetonitrile. The RPC fractions were then analyzed by both matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS). When this method was applied to a study of lymphosarcoma in canines, it was found that during chemotherapy, a series of fucosylated proteins in the blood of patients decreased in concentration more than 2-fold. Two of the proteins identified, CD44 and E-selectin, are known to be involved in cell adhesion and cancer cell migration. The observed aberrant fucosylation of these proteins is consistent with the hypothesis that CD44 and E-selectin play a key role in metastasis and the spread of cancer cells to remote sites.

Amino Acid Sequence↗
PubMed2026

Urine and Serum Proteome and Lipidome Analysis of Naturally Aging Feline Species.

Aging in companion animals such as cats closely relates to human aging in environmental exposures and disease manifestation, providing a valuable model for identifying biomarkers of age-associated decline. This study provides a combined proteomic and lipidomic analysis of serum and urine from naturally aging domestic cats aged 3.8-16 years, grouped as adult, old, and senior, to identify age-related molecular changes across biofluids. Label-free quantitative proteomics identified 901 urinary and 238 serum proteins, with 75 urinary proteins significantly altered with age that are linked to kidney disease, hypertension, neurodegeneration, and metabolic disorders. In contrast, only six serum proteins differed significantly between adult and old/senior cats, including decreased Apolipoprotein A-I (APOA1) in seniors, a protein linked with cognitive function in aging. Untargeted lipidomics revealed increases in specific serum triacylglycerols, phosphatidylcholines (PCs), and sphingomyelins, while urinary lipid profiles showed limited age-related changes, with some PCs decreasing, and diacylglycerols increasing with age. These results demonstrate distinct systemic and renal molecular remodeling during feline aging and highlight the utility of integrated omics analyses of biological fluids for identifying molecular alterations relevant to both feline and human aging.

Animals↗
PubMed2003

Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology.

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M(r) proteins and was complemented by the 2-DE/MS method, which showed a preference for low M(r) proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2024

Proteomic Heterogeneity of the Extracellular Matrix Identifies Histologic Subtype-Specific Fibroblast in Gastric Cancer.

Gastric cancer (GC) is a highly heterogeneous disease regarding histologic features, genotypes, and molecular phenotypes. Here, we investigate extracellular matrix (ECM)-centric analysis, examining its association with histologic subtypes and patient prognosis in human GC. We performed quantitative proteomic analysis of decellularized GC tissues that characterizes tumorous ECM, highlighting proteomic heterogeneity in ECM components. We identified 20 tumor-enriched proteins including four glycoproteins, serpin family H member 1 (SERPINH1), annexin family (ANXA3/4/5/13), S100A family (S100A6/8/9), MMP14, and other matrisome-associated proteins. In addition, histopathological characteristics of GC reveals differential expression in ECM composition, with the poorly cohesive carcinoma-not otherwise specified (PCC-NOS) subtype being distinctly demarcated from other histologic subtypes. Integrating ECM proteomics with single-cell RNA sequencing, we identified crucial molecular markers in the PCC-NOS-specific stroma. PCC-NOS-enriched matrisome proteins and gene expression signatures of adipogenic cancer-associated fibroblasts (CAFadi) are closely linked, both associated with adverse outcomes in GC. Using tumor microarray analysis, we confirmed the CAFadi surface marker, ATP binding cassette subfamily A member 8 (ABCA8), predominantly present in PCC-NOS tumors. Our ECM-focused analysis paves the way for studies to determine their utility as biomarkers for patient stratification, offering valuable insights for linking molecular and histologic features in GC.

Humans↗
PubMed2006

Selective isolation of lysine-free tryptic peptides delimited by arginine residues: A new tool for proteome analysis.

Tryptic digestion of biotinylated Lys-C peptides followed by affinity chromatography allows the selective isolation of lysine-free tryptic peptides delimited by arginine residues (RRnK peptides). In silico analysis revealed that RRnK peptides represent 87% of the whole proteomes and their specific isolation simplifies the complex peptide mixture (5 peptides per protein). The good recoveries and high selectivity obtained in the isolation of RRnK peptides anticipate the applicability of this method in 2DE-free quantitative proteome analyses.

Amino Acid Sequence↗
PubMed2003

A novel proteomic screen for peptide-protein interactions.

Regulated interactions between short, unstructured amino acid sequences and modular protein domains are central to cell signaling. Here we use synthetic peptides in "active" (e.g. phosphorylated) and "control" (e.g. non-phosphorylated) forms as baits in affinity pull-down experiments to determine such interactions by quantitative proteomics. Stable isotope labeling by amino acids in cell culture distinguishes specific binders directly by the isotope ratios determined by mass spectrometry (Blagoev, B., Kratchmarova, I., Ong, S.-E., Nielsen, M., Foster, L. J., and Mann, M. (2003) Nat. Biotechnol. 21, 315-318). A tyrosine-phosphorylated peptide of the epidermal growth factor receptor specifically retrieved the Src homology domain (SH) 2- and SH3 domain-containing adapter protein Grb2. A proline-rich sequence of Son of Sevenless also specifically bound Grb2, demonstrating that the screen maintains specificity with low affinity interactions. The proline-rich Sos peptide retrieved only SH3 domain containing proteins as specific binding partners. Two of these, Pacsin 3 and Sorting Nexin 9, were confirmed by immunoprecipitation. Our data are consistent with a change in the role of Sos from Ras-dependent signaling to actin remodeling/endocytic signaling events by a proline-SH3 domain switch.

Actins↗
PubMed2004

The use of isotope-coded affinity tags (ICAT) to study organelle proteomes in Arabidopsis thaliana.

Organelle proteomics is the analysis of the protein contents of a subcellular compartment. Proteins identified in subcellular proteomic studies can only be assigned to an organelle if there are no contaminants present in the sample preparation. As a result, the majority of plant organelle proteomic studies have focused on the chloroplast and mitochondria, which can be isolated relatively easily. However, the isolation of components of the endomembrane system is far more difficult due to their similar sizes and densities. For this reason, quantitative proteomics methods are being developed to enable the assignment of proteins to a specific component of the endomembrane system without the need to obtain pure organelles.

Arabidopsis↗
PubMed2004

Proteogenomic mapping as a complementary method to perform genome annotation.

The accelerated rate of genomic sequencing has led to an abundance of completely sequenced genomes. Annotation of the open reading frames (ORFs) (i.e., gene prediction) in these genomes is an important task and is most often performed computationally based on features in the nucleic acid sequence. Using recent advances in proteomics, we set out to predict the set of ORFs for an organism based principally on expressed protein-based evidence. Using a novel search strategy, we mapped peptides detected in a whole-cell lysate of Mycoplasma pneumoniae onto a genomic scaffold and extended these "hits" into ORFs bound by traditional genetic signals to generate a "proteogenomic map". We were able to generate an ORF model for M. pneumoniae strain FH using proteomic data with a high correlation to models based on sequence features. Ultimately, we detected over 81% of the genomically predicted ORFs in M. pneumoniae strain M129 (the originally sequenced strain). We were also able to detect several new ORFs not originally predicted by genomic methods, various N-terminal extensions, and some evidence that would suggest that certain predicted ORFs are bogus. Some of these differences may be a result of the strain analyzed but demonstrate the robustness of protein analysis across closely related genomes. This technique is a cost-effective means to add value to genome annotation, and a prerequisite for proteome quantitation and in vivo interaction measures.

Chromosome Mapping↗
PubMed2026

Differential Proteomic Profiling of Responders and Non-responders to Direct-Acting Antivirals Treatment in Chronic Hepatitis C Virus Infection.

Hepatitis C Virus (HCV), particularly genotype 3 (GT-3), is highly prevalent in India and is associated with faster progression to cirrhosis, hepatocellular carcinoma, and higher treatment failure rates. Although Direct-Acting Antivirals (DAAs) have revolutionized HCV therapy, 5-10% of patients fail to achieve sustained virological response (SVR). This proteomic study aimed to identify changes in the proteomic profile before and after treatment of both responders and non-responders to HCV treatment. Paired plasma samples from HCV GT-3 infected patients were collected before and 12 weeks after initiating DAAs treatment, along with healthy controls. Quantitative proteomic analysis was performed on the paired samples. Differentially expressed proteins (DEPs) were identified and subjected to functional analysis including gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) network analysis. GSEA revealed enrichment in extracellular matrix organization and innate immune pathways. Expression patterns of candidate proteins selected based on fold change and false discovery rate (FDR) criteria were further evaluated in an independent cohort. Western blot confirmed key expression trends of candidate proteins. Proteins linked to extracellular matrix remodeling and angiogenesis showed differential expression patterns. Successful validation of these candidate proteins in large independent cohorts holds potential to predict therapeutic outcomes.

Humans↗