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PubMed2026

Ligand-Mediated Reprogramming Redirects Liver-Tropic Ionizable Lipid Nanoparticles for Lung-Selective mRNA Delivery.

Systemic delivery of messenger RNA (mRNA) to target tissues and cells using lipid nanoparticles (LNPs) holds transformative potential for gene therapy. However, most clinically validated LNP exhibit strong liver tropism, and redirecting their organ specificity without redesigning entirely new chemistries remains challenging. Here we present a ligand-mediated lipid reprogramming approach that repurposes chemically defined, liver-tropic, ionizable lipids (lipidoids) for mRNA delivery beyond the liver. From a library of 90 degradable lipidoids, we identified 2-t6b as a potent liver-targeting platform. By site-specific displaying of small molecule ligands onto 2-t6b headgroup, we engineered a series of reconfigured lipidoids that achieve lung-specific targeting while retaining the parent delivery scaffold. Ligand7-2-t6b-lipid-functionalized LNP achieved over 200-fold higher mRNA translation in the lungs compared to the parent liver-tropic LNP. Proteomics and molecular docking analysis revealed enhanced binding of the modified lipid to vitronectin, a serum glycoprotein that improves integrin binding and thus promotes cellular uptake and translation efficiency. Ligand-mediated 2-t6b/ligand7 LNPs achieved outperformed efficacy and therapeutic potential in lung-specific genome editing relative to SORT-constructed 2-t6b LNP system. Our modular reprogramming strategy provides a generalizable framework to upgrade existing liver-biased LNPs into lung-selective mRNA carriers, advancing next-generation tissue-specific mRNA therapies for gene editing, protein replacement therapy, and regenerative medicine.

RNA, Messenger↗

PubMed2003

Combined use of proteomic analysis and enzyme activity assays for metabolic pathway analysis of glycerol fermentation by Klebsiella pneumoniae.

The fed-batch fermentation of glycerol to 1,3-propanediol by Klebsiella pneumoniae displayed an unusual dynamic behavior that can be clearly divided into four distinct phases according to cell growth and CO(2) evolution rate. Metabolism changed significantly during the different phases as reflected by the varied specific rates of substrate consumption and product formation. An assay of activities of the three initial enzymes of glycerol metabolism, namely glycerol dehydratase (GDHt), glycerol dehydrogenase (GDH), and 1,3-propanediol-oxidoreductase (PDOR), showed apparently different patterns of expression. To understand the culture dynamics and patterns of enzyme formation at a more systemic level we analyzed the expression patterns of intracellular proteins of K. pneumoniae from different phases of the fed-batch fermentation using two-dimensional gel electrophoresis (2DE). Two new enzymes, namely a phosphoenolpyruvate-dependent dihydroxyacetone kinase (DHAK II) and a hypothetical oxidoreductase (HOR), which are directly related to glycerol metabolism and 1,3-propanediol formation, were identified among the highly expressed proteins. The changes in expression of these new enzymes and several other proteins identified from the 2DE analysis helped to understand not only the dynamic behavior of the fed-batch fermentation reported in this work but also some previously insufficiently understood phenomena related to this fermentation process. In particular, we demonstrated the combined use of proteomic analysis and enzyme activity assay data for metabolic pathway analysis and for a better identification of targets for bioprocess improvement.

Amino Acid Sequence↗

PubMed2003

Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysis.

Isoelectric focusing (IEF) separations, in general, involve the use of the entire channel filled with a solution mixture containing protein/peptide analytes and carrier ampholytes for the creation of a pH gradient. Thus, the preparative capabilities of IEF are inherently greater than most microfluidics-based electrokinetic separation techniques. To further increase sample loading and therefore the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from solution reservoirs, is demonstrated in this study. The proteins/peptides continuously migrate into the plastic microchannel and encounter a pH gradient established by carrier ampholytes originally present in the channel for focusing and separation. Dynamic sample introduction and analyte focusing in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Under the influence of 30 min electrokinetic injection at constant electric field strength of 500 V/cm, the sample loading is enhanced by approximately 10-100 fold in comparison with conventional IEF.

Animals↗

PubMed2005

A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in Escherichia coli cells.

An important consideration in the measurement of quantitative changes in protein expression is the consistency of the observations for a given technique as well as the reproducibility of the experiment. A quantitative assessment of the technical and biological variability is crucial to avoid erroneous inferences and conclusions. Two methods for measuring quantitative changes in protein expression are two-dimensional electrophoresis (2-DE) and shotgun proteomics of isobaric-tagged samples using iTRAQ reagents. An assessment of changes in Escherichia coli protein expression in response to rhsA induction demonstrates that half of the quantified protein expression ratios have a coefficent of variation (CV) less than 0.31 using 2-DE and less than 0.24 using isobaric tags; whereas 95% of the quantified protein expression ratios have a CV less than 0.81 using 2-DE and less than 0.53 using isobaric tags. The selective removal of outlier data points from the shotgun method using Grubb's and Rosner's statistical outlier tests improves the consistency of the quantitation data obtained.

Chromatography, Liquid↗

PubMed2005

Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples.

Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.

Blood Proteins↗

PubMed2005

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome.

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.

Amino Acid Sequence↗

PubMed2004

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry.

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.

Amino Acid Sequence↗

PubMed2026

Multidimensional Protein Corona Analysis Toward Predictive Nano-Bio Interface Design.

Nanoparticles entering biological fluids are rapidly coated by proteins and other biomolecules, converting their synthetic surfaces into biologically active nano-bio interfaces. These coronas regulate colloidal stability, immune recognition, cellular uptake, biodistribution, pharmacokinetics, cargo delivery, and toxicity. Yet a protein list obtained by mass spectrometry captures only part of this interface. Corona identity and function are also shaped by protein organization, binding stability, exchange dynamics, conformational changes, and molecular accessibility. Here, we discuss recent progress in protein corona isolation and analysis from a question-oriented analytical perspective, with emphasis on how centrifugation, magnetic recovery, affinity- or chemistry-enabled capture, chromatography, filtration, and field-flow fractionation (FFF) influence the fidelity, integrity, and comparability of recovered coronas. We then examine how proteomic profiling can be integrated with binding measurements, interfacial structural analysis and functional validation to distinguish descriptive corona signatures from biologically meaningful mechanisms. We further consider how biofluid composition, disease state, tissue interfaces and cellular environments remodel corona identity, presentation, and bioactivity. Finally, we argue that standardized reporting, computational modeling, and AI-enabled approaches are essential for converting protein corona datasets into reproducible and predictive knowledge that can guide the design of drug delivery systems and precision nanomedicines.

Protein Corona↗

PubMed2003

Epitope-targeted proteome analysis: towards a large-scale automated protein-protein-interaction mapping utilizing synthetic peptide arrays.

We describe the development of a process for the genome-wide mapping of interactions between protein domains and peptide ligands entirely based on high-throughput biochip technologies. A phage library displaying protein domains from a randomly fragmented and cloned cDNA library will be "panned" on an array of synthetic peptide ligands. After multiplexed affinity enrichment, peptide-specific phage populations will be automatically eluted, propagated, labelled and identified by hybridisation to a DNA microarray. Peptide arrays are synthesized in situ by SPOT synthesis on a planar substrate. By utilizing a commercially available library of human brain cDNA plus a set of distinct model domains cloned into T7-phage, we could show that a single panning round on an array of known peptide ligands for these model domains synthesized on a cellulose membrane can yield an enrichment of better than a factor of 1,000. This is sufficient to detect peptide-specific enrichment of Cy3(post-panning) against Cy5(pre-panning)-labelled phage DNA inserts on a cDNA microarray. Thus, the proof-of-principle of our approach could be successfully demonstrated and first interaction data are being collected.

Amino Acid Sequence↗

PubMed2005

Spectroscopic imaging of protein crystals in crystallization drops.

Automatic imaging and scoring of crystallization drops is an essential step in high-throughput crystallography. Presently, white-light images of crystallization drops are acquired robotically and the images are analyzed and scored using pattern recognition algorithms. However, the scoring part remains unreliable as crystals and microcrystals are not always recognized by existing feature-extraction and recognition algorithms. We propose a fundamental shift in crystal monitoring through spectroscopic imaging of crystallization drops. This method converts the problem of automatic crystal detection from one of pattern recognition into one of intensity (concentration) analysis. The latter can be more robust and reliable.

Crystallization↗

PubMed2005

Carbonylation and glutathionylation of proteins in the blue mussel Mytilus edulis detected by proteomic analysis and Western blotting: Actin as a target for oxidative stress.

Protein expression profiles (PEPs) were generated by two-dimensional electrophoresis (2-D SDS-PAGE) for gill and digestive glands of Mytilus edulis sampled from a polluted and reference site in Cork Harbour, Ireland. Similar patterns and expression levels were found for both sites in silver stained gels. However, Western blotting for carbonylated proteins demonstrated higher levels of specific carbonylation of proteins in tissues from animals in the polluted site. Animals from the reference site were acclimated in holding tanks, exposed to 1 mM H2O2 for 24 h, dissected and analysed by 2-D SDS-PAGE. Again, generally similar PEPs were found in control and exposed animals for gill and digestive gland but carbonylation was more pronounced in polluted and exposed animals. Western blotting of extracts after one-dimensional electrophoresis with antibodies to glutathione and actin revealed that gill proteins are glutathionylated more strongly than digestive gland and that this process is more pronounced in polluted animals than in controls. We conclude that carbonylation and glutathionylation can occur in gill and digestive gland in response to oxidative stress in M. edulis. Actin is a major target for both glutathionylation and carbonylation under oxidative stress conditions.

Actins↗

PubMed2005

Protein carbonylation and heat shock response in Ruditapes decussatus following p,p'-dichlorodiphenyldichloroethylene (DDE) exposure: a proteomic approach reveals that DDE causes oxidative stress.

Protein carbonylation and levels of heat shock proteins (hsp; 60, 70 and 90 kDa) were measured in gill, mantle and digestive gland of Ruditapes decussatus following exposure to p,p'-dichlorodiphenyldichloroethylene (DDE). Heat shock response was measured by immunoblotting using antibodies specific to heat shock proteins (hsps). Densitometry analysis of individual bands revealed no difference between control and treated samples except appearance of hsp90 in DDE-treated mantle. Carbonylated protein content was determined following 2,4-dinitrophenylhydrazine derivatization and two-dimensional electrophoresis coupled with western blotting. Immunoblotting with dinitrophenol-specific antibody revealed extensive differences in both extent and number of carbonylated proteins in mantle and digestive gland in response to DDE while gill was unaffected. These results demonstrate for the first time that DDE causes tissue-specific formation of reactive oxygen species in clams.

Animals↗

PubMed2006

Protein responses in blue mussels (Mytilus edulis) exposed to organic pollutants: a combined CYP-antibody/proteomic approach.

Polyclonal antibodies were raised against highly conserved, trans-metazoan sequences of cytochrome P450 (CYP) families 2 and 4 and used to investigate responses in the common blue mussel (Mytilus edulis) exposed to various organic contaminants. The results were evaluated by means of cross-reacting proteins on Western blots of both one- and two-dimensional electrophoresis gels, and by scanning spectroscopy measurements of total CYP content. Furthermore, a proteomic approach was applied aimed at elucidating exposure-related protein changes in a more general term. Identities of isolated proteins were searched by means of peptide mass fingerprints obtained from MALDI-TOF MS analyses. The results demonstrated that both antibodies rendered several cross-reactive bands when probed on Western blots. The most obvious cross-reaction of the CYP2 antibody was with a strongly expressed protein of size approximately 57kDa, pI 4.5-4.6, whereas the CYP4 antibody cross-reacted with a protein of size approximately 55kDa, pI 5.6. However, expression of cross-reacting proteins did not change as a result of the exposures, and resulted only in small and insignificant fluctuations in total CYP content. As a contrast, silver-stained 2DE gels showed that several microsomal proteins were affected in individuals exposed to diallylphthalate as well as crude oil, with and without a spike of alkylphenols and PAHs. Mass spectrometry based analyses of excised, trypsin-digested spots did so far not decipher the identities of the proteins affected by the exposures, nor of those cross-reacting with CYP2 and CYP4 antibodies. This study has underlined the power of the proteomic approach in environmental toxicology, although protein identification was not successful. The missing identities of the proteins cross-reacting with the CYP2- and CYP4-antibodies does not enable a clear conclusion as to whether or not these peptides actually represent CYP iso-enzymes.

Amino Acid Sequence↗

PubMed2004

Proteome analysis of mouse macrophages treated with anthrax lethal toxin.

Anthrax toxin produced by Bacillus anthracis is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF into the cytosol. EF is a calmodulin-dependent adenylate cyclase that causes edema whereas LF is a zinc metalloprotease and leads to necrosis of macrophages. It is also important to note that the exact mechanism of LF action is still unclear. With this view in mind, in the present study, we investigated a proteome wide effect of anthrax lethal toxin (LT) on mouse macrophage cells (J774A.1). Proteome analysis of LT-treated and control macrophages revealed 41 differentially expressed protein spots, among which phosphoglycerate kinase I, enolase I, ATP synthase (beta subunit), tubulin beta2, gamma-actin, Hsp70, 14-3-3 zeta protein and tyrosine/tryptophan-3-monooxygenase were found to be down-regulated, while T-complex protein-1, vimentin, ERp29 and GRP78 were found to be up-regulated in the LT-treated macrophages. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation proteins play an important role in the LT-mediated macrophage cell death.

Adenosine Triphosphate↗

PubMed2005

Development of a sample preparation method for fungal proteomics.

Since filamentous fungi including basidiomycetous fungi possess an exceptionally robust cell wall as in microorganisms, effective extraction of intracellular proteins is a key step for fungal proteomic studies. To overcome the experimental obstacle caused by cell walls, we utilized fungal protoplasts, prepared from the brown-rot basidiomycete, Tyromyces palustris. The amount and quality of proteins extracted from the protoplast cells were much higher than that from the mycelial cells. Quantitative comparisons of proteome maps prepared from mycelial and protoplast cells indicated protein spots with a wider range of molecular weights and pIs in the protoplast sample. Furthermore, no streaking or tailing was observed in the protoplasts, suggesting that effective extraction of intracellular proteins from protoplasts might help suppress degradation of proteins during this process. In addition to the efficiency of protein extraction, simple and efficient subcellular fractionation was also achieved using protoplast cells.

Basidiomycota↗

PubMed2005

Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites.

Protein profiles of two isolates of Neospora caninum (KBA-2 and JPA1) and Toxoplasma gondii RH strain were investigated by proteomic approach. Approximately, 78% of protein spots on two-dimensional gel electrophoresis (2-DE) profiles and 80% of antigen spots on 2-DE immunoblotting profiles were exhibited to share the same pI and M(r) between KBA-2 and JPA1 of N. caninum. On the other hand, a total of 30 antigen spots of T. gondii were recognized on 2-DE immunoblotting profile using rabbit antiserum against N. caninum KBA-2. A number of homologue proteins, such as heat shock protein 70, tubulin alpha- and beta-chain, putative protein disulfide isomerase, actin, enolase and 14-3-3 protein homologue are believed as the conserved proteins in both N. caninum and T. gondii. On the contrary, NcSUB1, NcGRA2 and NCDG1 (NcGRA7) might be the species-specific proteins for N. caninum tachyzoites. The present study showed that the high degree of similarity between N. caninum isolates (KBA-2 and JPA1), whereas large differences between N. caninum and T. gondii were noticed by proteome comparisons.

Animals↗

PubMed2004

Proteomic fingerprinting of HIV-1-infected human monocyte-derived macrophages: a preliminary report.

Mononuclear phagocytes (MP; blood monocytes, alveolar, lymph node, and brain macrophages and microglia) are vehicles for dissemination and principle target cells for human immunodeficiency virus type 1 (HIV-1) infection. Notably, viral persistence in macrophages occurs despite ongoing phagocytic, intracellular killing, innate and adaptive immune responses. To assess potential pathways for how HIV-1 may bypass antiviral MP responses, we used proteomic tests to evaluate protein fingerprints of HIV-1-infected human monocyte-derived macrophages 7 days after viral infection. By using weak cation exchange chips, 58 proteins were found up- or down-regulated after HIV-1(ADA) infection. Several of these proteins were identified by microsequencing. It is probable that cellular proteins identified by proteomic fingerprinting could assist in unraveling how persistent viral infection occurs in MP lineage cells. Moreover, this evolving technology can be utilized to unravel changes in immune activities initiated by interactions between virus, environmental cues and drugs of abuse.

Cells, Cultured↗

PubMed2004

The genomics of malaria infection.

Malaria research is now dominated by information flowing from the genome sequencing projects and the associated transcriptome- and proteome-mapping projects. As more species are sequenced, comparative and phylogenetic comparisons are improving the quality of gene finding, and are providing various approaches to the identification of genes important to parasite biology and the pathogenesis of disease. We are still in the early days of exploiting these data in a systematic way and the sheer volume of data presents daunting challenges. This article reviews the progress in using this genomic information and discusses opportunities for other approaches.

Animals↗