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PubMed2006

Reverse genetics for proteomics: from proteomic discovery to scientific content.

Proteomic strategies generally result in rather descriptive outcomes. Although proteomic patterns may lead to understanding of cellular function or failure, they do not provide insights into cellular mechanisms of normal physiology or molecular mechanisms of disease. The integration of single events or patterns into complex biological processes and signaling networks must be functionally validated by studying these processes in a dynamic physiological context. Effective methodologies, applied at the level of intact cells and model organisms that monitor their function, topology and interaction of proteins are necessary and essential. Reverse genetic strategies can greatly advance the understanding of patterns identified through proteomic analysis. RNAi as an accessible method for routine functional analysis of cells and whole model organisms represents a quantum leap in revolutionizing reverse genetics. This paper aims at describing an integrated workflow linking proteome-based discovery to molecular analysis of single genes and proteins by RNAi, potentially contributing to the advanced understanding of molecular mechanisms in neurodegenerative disease, including Parkinson's disease.

Animals↗

PubMed2005

Gene and protein expression profiling of the microvascular compartment in experimental autoimmune encephalomyelitis in C57Bl/6 and SJL mice.

Dysfunction of the blood-brain barrier (BBB) is a hallmark of inflammatory diseases of the central nervous system (CNS) such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). The molecular mechanisms leading to BBB breakdown are not well understood. In order to find molecules involved in this process, we used oligonucleotide microarrays and proteomics to analyze gene and protein expression of the microvascular compartment isolated from brains of C57Bl/6 and SJL/N mice afflicted with EAE and the microvascular compartment isolated from healthy controls. Out of the 6500 known genes and expressed sequence tags (ESTs) studied, expression of 288 genes was found to be changed. Of these genes 128 were altered in the microvascular compartment in both EAE models. Six proteins were identified to be present at altered levels. In addition to the expected increased expression of genes coding for molecules involved in leukocyte recruitment, genes not yet ascribed to EAE pathogenesis were identified. Thus, proteomics and gene array screens of the microvascular compartment are valid approaches, that can be used to define novel candidate molecules involved in EAE pathogenesis at the level of the BBB.

Animals↗

PubMed2026

Comparative Multiomics Analysis of Cerebral Organoid-Derived Exosomes during Organoid Maturation.

Cerebral organoids derived from human pluripotent stem cells recapitulate key features of early brain development and provide a physiologically relevant model for neurogenesis. Exosomes secreted by these organoids carry bioactive cargo and offer a noninvasive means to monitor maturation and intercellular communication. We performed comprehensive multiomics profiling of exosomes collected from cerebral organoids at defined developmental stages to evaluate their utility as biomarkers of neuronal differentiation. Metabolomic analysis revealed a progressive decline in amino acids, including glutamic acid, consistent with increased metabolic demand during neurogenesis. Lipidomic and neurosteroid profiling showed dynamic increases in phosphatidylethanolamine and pregnenolone, reflecting synaptic membrane formation and signaling. Transcriptomic and proteomic analyses identified stage-specific neurodevelopmental signatures, with key markers mirroring those of parent organoids. Collectively, cerebral organoid-derived exosomes faithfully reflect organoid maturation and provide a robust platform for tracking in vitro brain development.

Humans↗

PubMed2026

Development of a High-Sensitivity Glycoproteomics Approach for Fc-Specific Quantification of IgG Core Fucosylation in Traumatic Brain Injury.

Traumatic brain injury (TBI) triggers complex neuroinflammatory cascades that involve sustained immune activation and dysregulated antibody effector functions. Immunoglobulin G (IgG) Fc N-glycosylation, particularly core fucosylation, critically modulates immune signaling through altered Fcγ receptor (FcγR) interactions; however, its role in TBI remains unexplored. Here, we developed a high-sensitivity, mass spectrometry-based glycoproteomics method for the systematic analysis of IgG Fc core fucosylation dynamics following TBI. The approach integrates Fc-specific enzymatic truncation with GlycINATOR (EndoS2) and tryptic digestion, followed by high-resolution LC-MS/MS profiling, enabling confident identification of truncated Fc glycopeptides. Furthermore, a targeted parallel reaction monitoring (PRM) strategy allowed direct quantification of core fucosylated and afucosylated glycopeptides from 10 μg of crude serum protein, eliminating the need for IgG purification. Our results reveal time-dependent and subclass-specific remodeling of IgG Fc fucosylation postinjury, characterized by an overall reduction in fucosylated species and a relative increase in afucosylation. Collectively, this study establishes a scalable analytical platform for Fc-specific glycosylation profiling and identifies IgG core fucosylation as a candidate molecular indicator of immune dysregulation in TBI, providing new insights into post-traumatic immune regulation.

Brain Injuries, Traumatic↗

PubMed2006

Proteomic analysis of rat hippocampal plasma membrane: characterization of potential neuronal-specific plasma membrane proteins.

The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively proteins of hippocampal plasma membrane (PM) and detect the neuronal-specific PM proteins, we performed a proteomic analysis of rat hippocampus PM using the following three technical strategies. First, proteins of the PM were purified by differential and density-gradient centrifugation from hippocampal tissue and separated by one-dimensional electophoresis, digested with trypsin and analyzed by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS). Second, the tryptic peptide mixture from PMs purified from hippocampal tissue using the centrifugation method was analyzed by liquid chromatography ion-trap ESI-MS/MS. Finally, the PM proteins from primary hippocampal neurons purified by a biotin-directed affinity technique were separated by one-dimensional electrophoresis, digested with trypsin and analyzed by ESI-Q-TOF-MS/MS. A total of 345, 452 and 336 non-redundant proteins were identified by each technical procedure respectively. There was a total of 867 non-redundant protein entries, of which 64.9% are integral membrane or membrane-associated proteins. One hundred and eighty-one proteins were detected only in the primary neurons and could be regarded as neuronal PM marker candidates. We also found some hypothetical proteins with no functional annotations that were first found in the hippocampal PM. This work will pave the way for further elucidation of the mechanisms of hippocampal function.

Animals↗

PubMed2006

The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function.

Upon injury, astrocytes assume an activated state associated with the release of inflammatory mediators. To model this, we stimulated murine primary astrocytes with a complete inflammatory cytokine mix consisting of TNF-alpha, IL-1beta and IFN-gamma. We analysed the transcriptional response of 480 genes at 4 and 16 h after stimulation on a chip designed to give a representative overview over the inflammation-relevant part of the transcriptome of macrophage-like cells. The list of the 182 genes found to be significantly regulated in astrocytes revealed an intriguing co-ordinate regulation of genes linked to the biological processes of antiviral/antimicrobial defence, antigen presentation and facilitation of leucocyte invasion. The latter group was characterized by very high up-regulations of chemokine genes. We also identified regulations of a thymidylate kinase and an interferon-regulated protein with a tetratricopeptide motive, both up to now only known from macrophages. The transcriptional regulations were confirmed on the protein level by a proteomic analysis. These findings taken together suggest that activated astrocytes in brain behave similarly in many respects to inflamed macrophages in the periphery.

Animals↗

PubMed2006

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques.

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.

Animals↗

PubMed2007

Crystallins in the eye: Function and pathology.

Crystallins are the predominant structural proteins in the lens that are evolutionarily related to stress proteins. They were first discovered outside the vertebrate eye lens by Bhat and colleagues in 1989 who found alphaB-crystallin expression in the retina, heart, skeletal muscles, skin, brain and other tissues. With the advent of microarray and proteome analysis, there is a clearer demonstration that crystallins are prominent proteins both in the normal retina and in retinal pathologies, emphasizing the importance of understanding crystallin functions outside of the lens. There are two main crystallin gene families: alpha-crystallins, and betagamma-crystallins. alpha-crystallins are molecular chaperones that prevent aberrant protein interactions. The chaperone properties of alpha-crystallin are thought to allow the lens to tolerate aging-induced deterioration of the lens proteins without showing signs of cataracts until older age. alpha-crystallins not only possess chaperone-like activity in vitro, but can also remodel and protect the cytoskeleton, inhibit apoptosis, and enhance the resistance of cells to stress. Recent advances in the field of structure-function relationships of alpha-crystallins have provided the first clues to their underlying roles in tissues outside the lens. Proteins of the betagamma-crystallin family have been suggested to affect lens development, and are also expressed in tissues outside the lens. The goal of this paper is to highlight recent work with lens epithelial cells from alphaA- and alphaB-crystallin knockout mice. The use of lens epithelial cells suggests that crystallins have important cellular functions in the lens epithelium and not just the lens fiber cells as previously thought. These studies may be directly relevant to understanding the general cellular functions of crystallins.

Animals↗

PubMed2006

Characterization of the localization and function of NECAP 1 in neurons.

NECAPs (adaptin ear-binding clathrin-associated protein) are a new family of clathrin accessory proteins identified through a proteomic analysis of clathrin-coated vesicles (CCVs) from the brain. One member of this family, NECAP 1, is found primarily in tissues from the central nervous system and has been shown to be complexed tightly with a substantial portion of adaptor protein-2 (AP-2) in brain extracts. However, the function and intracellular location of this protein is unknown. In this study, we find that endogenous and epitope-tagged NECAP 1 co-localizes well with clathrin and AP-2 in punctate structures, many of which also contain the presynaptic markers synaptophysin, synaptotagmin or synaptic vesicle protein 2 (SV2). NECAP 1 was also detected by western blot in synaptic vesicle preparations. Overexpression of a truncation mutant of NECAP 1 (BC-NECAP 1) in neurons inhibited transferrin endocytosis but not epidermal growth factor (EGF) endocytosis, and this inhibition was dependent on an AP-2-binding WVQF motif. Moreover, overexpression of BC-NECAP 1 results in inhibition of synaptotagmin endocytosis both in unstimulated neurons and in neurons stimulated with potassium chloride. This inhibition was abrogated by truncation of the WVQF domain. We conclude from these observations that NECAP 1 plays a role in clathrin-mediated neuronal endocytosis, including a role in presynaptic endocytosis.

Adaptor Protein Complex 2↗

PubMed2005

Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies.

Malignant gliomas (astrocytomas) are lethal tumors that invade the brain. Invasive cell migration is initiated by extension of pseudopodia into interstitial spaces. In this study, U87 glioma cells formed pseudopodia in vitro as cells pushed through 3 microm pores of polycarbonate membranes. Harvesting pseudopodia in a novel two-step method provided material for proteomic analysis. Differences in the protein profiles of pseudopodia and whole cells were found using differential gel electrophoresis (DIGE) and immunoblotting. Proteins from two-dimensional (2D) gels with M(R)'s of 20-100 kDa and pI's of 3.0-10.0 were identified by peptide mass fingerprinting analysis using mass spectrometry. For DIGE, lysates of pseudopodia and whole cells were each labeled with electrophilic forms of fluorescent dyes, Cy3 or Cy5, and analyzed as mixtures. Analysis was repeated with reciprocal labeling. Differences in protein distributions were detected by manual inspection and computer analysis. Topographical digital maps of the scanned gels were used for algorithmic spot matching, normalization of background, quantifying spot differences, and elimination of artifacts. Pseudopodial proteins in Coomassie-stained 2D gels included isoforms of glycolytic enzymes as the largest group, seven of 24 proteins. Peptide mass fingerprint analysis of DIGE gels demonstrated increased isoforms of annexin (Anx) I, AnxII, enolase, pyruvate kinase, and aldolase, and decreased mitochondrial manganese superoxide dismutase and transketolase in pseudopodia. Specific antibodies showed restricted immunoreactivity of the hepatocyte growth factor (HGF) alpha chain to pseudopodia, indicating localization of its active form. Met (the HGF receptor), actin, and total AnxI were increased in pseudopodial lysates on immunoblots. Increased constituents of the pseudopodial proteome in glioma cells, identified in this study as actin, HGF, Met, and isoforms of AnxI, AnxII, and several glycolytic enzymes, represent therapeutic targets to consider for suppression of tumor invasion.

Annexin A1↗

PubMedDate not supplied

Building a foundation for the human proteome: the role of the Human Proteome Organization.

Proteomics holds both substantial promise and substantial challenges. For proteomics to bear fruit on a large scale from a disease investigation point of view, it is essential to build a solid foundation for the field. Given the magnitude of the challenges, it is necessary to build a foundation by bringing together the private and public sectors. The Human Proteome organization is promoting the field of proteomics by engaging in such an effort and is developing several major initiatives.

Antibodies↗

PubMed2025

SynchroSep-MS: Parallel LC Separations for Multiplexed Proteomics.

Achieving high throughput remains a challenge in MS-based proteomics for large-scale applications. We introduce SynchroSep-MS, a novel method for parallelized, label-free proteome analysis that leverages the rapid acquisition speed of modern mass spectrometers. This approach employs multiple liquid chromatography columns, each with an independent sample, simultaneously introduced into a single mass spectrometer inlet. A precisely controlled retention time offset between sample injections creates distinct elution profiles, facilitating unambiguous analyte assignment. We modified the DIA-NN workflow to effectively process these unique parallelized data, accounting for retention time offsets. Using a dual-column setup with mouse brain peptides, SynchroSep-MS detected approximately 16,700 unique protein groups, nearly doubling the peptide information obtained from a conventional single proteome analysis. The method demonstrated excellent precision and reproducibility (median protein %RSDs less than 4%) and high quantitative linearity (median R2 greater than 0.96) with minimal matrix interference. SynchroSep-MS represents a new paradigm for data collection and the first example of label-free multiplexed proteome analysis via parallel LC separations, offering a direct strategy to accelerate throughput for demanding applications such as large-scale clinical cohorts and single-cell analyses without compromising peak capacity or causing ionization suppression.

Proteomics↗

PubMed2005

Proteome analysis of mouse primary astrocytes.

Astrocytes play a role in energy metabolism, neuronal homeostasis and release of neuronal growth factors and several neurotransmitters. They also relate to a variety of brain diseases and contribute to restore brain dysfunction. Although current research has revealed several roles for astrocytes, knowledge on astrocytic protein expression is limited and a systematic and comprehensive proteome study of astrocytes has not been reported so far. We applied a proteomics technique based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) and unambiguously identified 301 spots corresponding to 191 individual proteins in primary mouse astrocytes. The identified proteins were from antioxidant, chaperone, cytoskeleton, nucleic acid binding, signaling, proteasomal, hypothetical and miscellaneous proteins. A reference database is provided and proteins were identified in astrocytes specifically and unambiguously for the first time. A reliable analytical tool independent of antibody availability and specificity along with tentative astrocytic marker proteins is described.

Animals↗

PubMed2026

Multi-omics-based study on the biological characteristics of kidney renal deficiency and blood stasis in ankylosing spondylitis.

OBJECIVE: To explore the objective biological evidence for the classification and diagnosis of Traditional Chinese Medicine (TCM) syndromes in ankylosing spondylitis (AS) using multiomics analysis. METHODS: Patients with AS were categorized into kidney deficiency and blood stasis syndrome (SX group) and damp-heat stasis syndrome (SR group). Transcriptomic sequencing and quantitative plasma proteomics were performed on patients with AS and healthy volunteers. Multiomics integration was used to characterize the biological basis of AS with renal deficiency and blood stasis syndrome. Specific proteins were validated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: Transcriptomic sequencing identified 31 significantly upregulated genes in patients with AS compared to healthy controls. These genes were primarily involved in tumor necrosis factor, interleukin-17, and nuclear factor kappa-B signaling pathways, as well as osteoblast differentiation and various viral infection pathways. Differentially expressed genes, including intercellular adhesion molecule 1 (ICAM1), 6-phosphofructo-2-kinase, cyclin-dependent kinase inhibitor 1A, interleukin 1 receptor antagonist, integrin alpha IIb, and myosin light chain 9 were more upregulated in the SX group than in the SR group. Quantitative proteomics identified 723 differential proteins associated with the disease and 788 differential proteins between the SX and SR groups. Notable proteins such as myeloperoxidase, cluster of differentiation 14, macrophage simulating 1 (MST1), and Ras homolog enriched in brain may serve as characteristic proteins of the SX group. By integrating transcriptomic and proteomic data, 45 associated differential molecules involved in platelet activation, pathogenic intestinal flora infection, glycolysis/gluconeogenesis, and T-cell receptor signaling pathways were identified in patients with AS compared to healthy controls. Additionally, ICAM1, MST1, C-X-C motif chemokine ligand 8 (CXCL8), suppressor of cytokine signaling 3 (SOCS3), and insulin-like growth factor binding protein 1 (IGFBP1) were detected in TCM syndromes by RT-qPCR and ELISA, showing upregulation in AS renal deficiency and blood stasis syndromes, which is consistent with the proteomic and transcriptomic results. CONCLUSIONS: ICAM1, MST1, CXCL8, SOCS3, and IGFBP1 were identified as biomarkers of renal deficiency and blood stasis syndrome in AS. This study provides a biological basis for the differential diagnosis of TCM syndromes in AS, offering new insights into Chinese medicine evidence and more precise Chinese medicine treatments for AS.

Humans↗

PubMed2004

Proteomic identification of specific oxidized proteins in ApoE-knockout mice: relevance to Alzheimer's disease.

We have examined oxidized proteins in the brain regions of wild-type (WT) and ApoE-knockout (KO) animals. Total protein oxidation in the hippocampus of young-KO (6 month) animals was approximately 2-fold greater than that of young-WT (6 month) animals and was similar to that of old-WT (18 month) and old-KO (18 month) animals. In the cortex of the same animals, the levels of total protein oxidation in all four groups were not significantly different. Two-dimensional electrophoresis (2-DE) coupled with immunostaining for protein carbonylation revealed six specific oxidation-sensitive proteins, the oxidation levels of which were increased in young-KO, old-WT, and old-KO mice compared with young-WT mice. These six oxidation-sensitive proteins were identified by mass spectrometry as glial fibrillary acidic protein, creatine kinase BB, disulfide isomerase, chaperonin subunit 5, dihydropyrimidase-related protein 2, and mortalin. These results indicate that the ApoE gene product offers protection against age-associated oxidative damage in the brain. Moreover, two of these proteins, creatine kinase and dihydropyrimidase-related protein 2, have recently been found to be oxidized in the brains of human subjects with Alzheimer's disease [Aksenov et al. J. Neurochem. 74: 2520-2527; 2000; Castegna et al. J. Neurochem. 82: 1524-1532; 2002]. These data suggest that the ApoE-knockout mouse serves as an appropriate model for studying pathogenic oxidative mechanisms influencing risk and progression of Alzheimer's disease.

Aging↗

PubMed2005

Ethanol and brain damage.

It is now well established that even uncomplicated alcoholics who have no specific neurological or hepatic problems show signs of regional brain damage and cognitive dysfunction. Improvements in neuroimaging technology, magnetic resonance imaging, magnetic resonance spectroscopy and positron emission tomography have contributed significantly, revealing alcoholic-specific changes in the CNS associated with neuropsychological abnormalities. Although greater efforts are needed, a human brain bank specifically targeting alcohol cases is now able to provide fresh and frozen tissue for alcohol researchers. These tissues can be used to test hypotheses developed using animal models and/or in vitro studies. The aim is to delineate mechanisms underlying alcohol-related brain damage in humans. The development of high-throughput, non-hypothesis-driven approaches using DNA microarrays and proteomics might also provide clues to this important problem.

Aged↗

PubMed2004

Demonstration of natural autoantibodies against the neurofilament protein alpha-internexin in sera of patients with endocrine autoimmunity and healthy individuals.

Serum anti-pituitary antibodies (APAs) to cytosolic antigens have been found in association with autoimmune hypophysitis, idiopathic hypopituitarism, and other autoimmune endocrinopathies. Here, an immunoblot method was used to search for serum autoantibody (AAb) reactivities against pituitary antigens, including nuclear and cytoskeletal proteins, in six patients with idiopathic hypopituitarism, 60 patients with type 1 diabetes, nine patients with autoimmune polyglandular syndrome (APS) type 1, and in 74 healthy controls. Frequent patient serum IgG reactivity was observed against a 60 kDa human pituitary antigen, and the cross-reactive 62 kDa protein from rat brain was identified as alpha-internexin (alpha-INX) by proteomic methods. IgG and IgM AAbs to this neuron-specific type IV intermediate filament (IF) protein were found in most sera of patients with endocrine autoimmunity as well as healthy subjects with no significant differences in frequencies between the groups, but the levels of IgM alpha-INX AAbs were higher in patients with hypopituitarism as compared to healthy controls (P = 0.032, Mann-Whitney U-test). These findings suggest that alpha-INX AAbs are not specifically related to autoimmune endocrine diseases and most probably are a part of the natural AAb repertoire. This is the first demonstration of alpha-INX AAbs as one of the predominant neuronal IF AAbs in human sera.

Adolescent↗

PubMed2005

Immunoaffinity reactors for prion protein qualitative analysis.

The cellular prion protein (PrPc) represents the substrate for generation of conformational aberrant PrP isoforms which occur in human and animal prion diseases. The published two-dimensional maps of human PrPc show a vast microheterogeneity of this glycoprotein. The main goal of this project was to develop a highly specific immunoaffinity reactor for qualitative analysis of PrP cellular isoforms isolated from brain homogenate, cerebrospinal fluid and other tissues. New techniques for affinity proteomics, carriers and immobilization chemistry were applied. The choice of matrix (chemical and magnetic properties, particle size and distribution, porosity) was the key factor that influenced the quality of the reactor and the nature of final applications. Mouse anti-prion IgGs directed to N-terminal and C-terminal epitopes (residues 23-40 and 147-165) were grafted in different manners to magnetic micro- and nanoparticles particularly developed for micro-CHIP application. High operational and storage stability of affinity reactors with minimized nonspecific absorption were achieved. The quality of the immunoreactors was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by two-dimensional electrophoresis.

Animals↗