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At least 145 records · Page 8Linked to original sources

PubMed2007

Effect of glycosylation on the protein pattern in 2-D-gel electrophoresis.

Single proteins, when analyzed with 2-D-PAGE, often show multiple spots due to PTMs. In gels of human body fluids, the spot patterns facilitate the assignment and identification of the proteins. We analyzed serums from patients with congenital disorders of glycosylation (CDG) in which glycoproteins are strongly impacted and exhibit highly distinguishable spot patterns compared to healthy controls. We detected a typical protein pattern for alpha1-acid glycoprotein (AGP) and transferrin (Trf) that are markers for CDG. AGP contains five glycosylation sites which results in a complex microheterogeneity of the glycoprotein. On the other hand, in Trf, a glycoprotein with only two glycosylation sites, mainly biantennary complex-type-N-linked glycans are bound. We used 2-D-PAGE, MALDI-TOF-MS, and ESI-MS for the analysis of these glycoproteins and their corresponding glycans. In AGP, the heterogenic glycosylation of the different glycosylation sites is responsible for the complex spot pattern. In contrast to AGP, the protein spots of Trf cannot be explained by glycosylation. We found strong evidence that oxidation of cysteine is responsible for the spot pattern. This study contradicts the commonly accepted assumption that the multiple protein spots of Trf observed in 2-D-PAGE are due, as in AGP, to the glycosylation of the protein.

Carbohydrate Metabolism, Inborn Errors↗

PubMed2007

Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF.

Biochemical studies of lipoproteins have shed light on their composition, highly contributing to the comprehension of their function. Due to the complexity of their structure, however, an in-depth structural analysis, in terms of components and PTMs, may still unravel important players in physiological and pathological processes of lipid metabolism. In this study, we performed a protein map of very low-density lipoprotein (VLDL) using a 2-DE MALDI-TOF/TOF proteomic approach. Several VLDL-associated apolipoproteins were identified, including five isoforms of apoE, three isoforms of apoC-IV, and one isoform each of apoC-III, apoM, apoA-I, and apoA-IV. Notably, we also identified seven isoforms of apoL-I and two isoforms of prenylcysteine lyase as new VLDL-associated proteins. Furthermore, we were able to identify PTM of apoE, which was found to be differently O-glycosylated at Thr212 residue, and PTM of apoL-I which we described, for the first time, to be phosphorylated at Ser296. While the physiological relevance of our finding remains to be assessed, we believe that our results will be useful as reference for future studies of VLDL structure in specific physiopathological conditions.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Identification of G2/M targets for the MAP kinase pathway by functional proteomics.

Although the importance of the extracellular signal-regulated kinase (ERK) pathway in regulating the transition from G1 to S has been extensively studied, its role during the G2/M transition is less well understood. Previous reports have shown that inhibition of the ERK pathway in mammalian cells delays entry as well as progression through mitosis, suggesting the existence of molecular targets of this pathway in M phase. In this report we employed 2-DE and MS to survey proteins and PTMs in the presence versus absence of MKK1/2 inhibitor. Targets of the ERK pathway in G2/M were identified as elongation factor 2 (EF2) and nuclear matrix protein, 55 kDa (Nmt55). Phosphorylation of each protein increased under conditions of ERK pathway inhibition, suggesting indirect control of these targets; regulation of EF2 was ascribed to phosphorylation and inactivation of upstream EF2 kinase, whereas regulation of Nmt55 was ascribed to a delay in normal mitotic phosphorylation and dephosphorylation. 2-DE Western blots probed using anti-phospho-Thr-Pro antibody demonstrated that the effect of ERK inhibition is not to delay the onset of phosphorylation controlled by cdc2 and other mitotic kinases, but rather to regulate a small subset of targets in M phase in a nonoverlapping manner with cdc2.

Amino Acid Sequence↗

PubMed2006

Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysis.

Liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS) has been used successfully for the characterization of biomolecules in proteomics in the last few years. This methodology relied largely on the use of reversed-phase chromatography, in particular C18-based resins, which are suitable for separation of peptides. Here we show that polymeric [polystyrene divinylbenzene] monolithic columns can be used to separate peptide mixtures faster and at a higher resolution. For 500 fmol bovine serum albumin, up to 68% sequence coverage and Mascot Mowse scores of >2000 were obtained using a 9 min gradient on a monolithic column coupled to an ion trap mass spectrometer with ultra-fast MS/MS scan rates. In order to achieve similar results using C18 columns, it was necessary to extend gradient times to 30 min. In addition, we demonstrate the utility of this approach for the analysis of whole Escherichia coli cell lysates by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) in combination with LC/MS/MS using 4 min gradients on monolithic columns. Our results indicate higher throughput capabilities of monolithic columns (3-fold gain in time or more) for conventional proteomics applications, such as protein identification and high sequence coverage usually required for detection of post-translational modifications (PTMs). Further optimization of sensitivity and quality of sequence information is discussed, in particular when combined with mass spectrometers that have very fast MS-MS/MS switching and scanning capabilities.

Animals↗

PubMed2025

ChIPmentation for Epigenomic Analysis in Fission Yeast.

Histone modifications and transcription factor-DNA interactions regulate vital processes such as transcription, recombination, repair, and accurate chromosome segregation. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been instrumental in studying genome-wide distribution of DNA-bound or chromatin-associated factors and histone posttranslational modifications (PTMs). Here, we describe a ChIPmentation protocol adapted for fission yeast, Schizosaccharomyces pombe. This method merges Tn5 mediated tagmentation with existing ChIP protocols, resulting in lower sample input requirements with significant reduction in hands-on time and sample preparation costs.

Schizosaccharomyces↗

PubMed2025

Genome-Wide Profiling of Histone Modifications in Fission Yeast Using CUT&Tag.

Eukaryotic DNA is organized in the nucleus in the form of chromatin. Nucleosomes, the fundamental unit of chromatin, are subject to many posttranslational modifications (PTMs) as well as compositional variations through incorporation of histone variants. These alterations play important roles in regulation of genome structure and activity. Genome-wide profiling of these regulatory features is essential for understanding of genome function. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) is a widely used method to assay genome-wide localization in fission yeast but suffers from the requirement for a large amount of input chromatin, antibodies, and a cumbersome experimental pipeline. New methods such as Cleavage Under Targets and Tagmentation (CUT&Tag), which combine the specificity of targeted cleavage and adapter insertion with the sensitivity of next-generation sequencing, enable identification and characterization of various epigenetic marks affording low input requirement as well as more streamlined protocols. However, these approaches have not been adapted for use in fission yeast, Schizosaccharomyces pombe. Here, we describe an adapted CUT&Tag protocol for epigenomic profiling in fission yeast using the heterochromatin-associated histone H3K9 methylation PTM for benchmarking.

Schizosaccharomyces↗

PubMed2025

Phosphorylation as a regulatory mechanism of HP1 protein multifunctionality.

The Heterochromatin Protein 1 (HP1) family proteins are key regulators of chromatin structure and genome function, acting as "reader" proteins that recognize and bind to histone H3 lysine 9 methylation (H3K9me). Beyond their canonical role in heterochromatin formation and transcriptional repression, HP1 proteins exhibit functional versatility, participating in transcriptional activation, RNA processing, DNA repair, and chromosome segregation. This multifunctionality is mediated partially by post-translational modifications (PTMs), with phosphorylation emerging as a central regulatory mechanism. This review explores the diverse effects of HP1 phosphorylation on protein function and chromatin interactions, focusing on Drosophila melanogaster HP1a and its orthologs, mammalian HP1α and S. pombe Swi6. Phosphorylation in the N-terminal tail enhances HP1's affinity for H3K9me, promoting transcriptional silencing. Mitotic phosphorylation of serine residues in the hinge region, regulated by kinases such as AURKB and NDR1/2, leads to chromatin release and relocalization to the kinetochore, enabling proper chromosome segregation. Additionally, phosphorylation modulates HP1 phase separation dynamics, influencing nuclear compartmentalization and chromatin condensation. These findings highlight phosphorylation as a versatile molecular switch that enables HP1 proteins to transition between structural and regulatory roles, contributing to their evolutionary conserved multifunctionality in genome regulation and cell division. Further investigation into HP1 phosphorylation across species and contexts is essential to fully understand its contributions to chromatin biology.

Phosphorylation↗

PubMed2003

Post-translational modifications of alpha-tubulin in Zea mays L are highly tissue specific.

To further understand post-translational modifications (PTMs) of plant alpha-tubulin, post-translationally modified alpha-tubulin isoforms from selected tissues of Zea mays L. were examined using two-dimensional electrophoresis and immunoblotting. Except for polyglycylated tubulin, tyrosinated, detyrosinated, acetylated and polyglutamylated alpha-tubulin isoforms were all present in maize tissues. Tyrosinated alpha-tubulin was the predominant variant in all cases, with isoforms alpha1-alpha4 (alpha5) being the most common components. Leaves exhibited a striking difference in PTM patterns of alpha-tubulin isoforms compared to other tissues examined. In leaves, several major specific isoforms were highly modified by detyrosination, acetylation and polyglutamylation. In pollen and anthers, only the most abundant isoform alpha3 was acetylated to an appreciable extent, and no acetylated isoform was found in roots. Similarly, in pollen, anthers and roots, only alpha3 was appreciably polyglutamylated. Additionally, a detyrosinated isoform alpha6 was present in anthers and in leaves, while the tyrosinated isoform alpha6 seemed to be pollen specific. These results indicate that certain types of PTM of plant alpha-tubulin preferentially occur in a tissue-specific way.

Electrophoresis, Polyacrylamide Gel↗

PubMed2004

Fish eggs as bioreactors: the production of bioactive luteinizing hormone in transgenic trout embryos.

We demonstrated the production of goldfish luteinizing hormone (gfLH) by the use of 4-day-old rainbow trout embryos as novel bioreactors. This expression system has several advantages: target proteins can be rapidly expressed at low cost, and recombinant proteins can be synthesized at low temperatures and can undergo complex post-translational modifications (PTMs). An expression vector containing gfLH cDNA was microinjected into fertilized trout eggs. After 4 days of incubation at 10 degrees C, transgenic embryos were harvested and glycosylated recombinant gfLH was recovered, which stimulated testosterone production in testicular fragments from the goldfish. This is the first report on the successful production of bioactive recombinant gonadotropin originated from cyprinid. Further, these results demonstrate that trout-embryo bioreactors are a potentially powerful tool for the production of functional recombinant proteins.

Animals↗

PubMed2006

Application of different fragmentation techniques for the analysis of phosphopeptides using a hybrid linear ion trap-FTICR mass spectrometer.

Electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) present complementary techniques for the fragmentation of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in addition to the commonly used collisionally activated dissociation (CAD). Both IRMPD and ECD have been shown to be applicable for an efficient sequencing of peptides and proteins, whereas ECD has proven especially valuable for mapping labile posttranslational modifications (PTMs), such as phosphorylations. In this work, we compare the different fragmentation techniques and MS detection in a linear ion trap and the ICR cell with respect to their abilities to efficiently identify and characterize phosphorylated peptides. For optimizing fragmentation parameters, sets of synthetic peptides with molecular weights ranging from approximately 1 to 4 kDa and different levels of phosphorylation were analyzed. The influence of spectrum averaging for obtaining high-quality spectra was investigated. Our results show that the fragmentation methods CAD and ECD allow for a facilitated analysis of phosphopeptides; however, their general applicability for analyzing phosphopeptides has to be evaluated in each specific case with respect to the given analytical task. The major advantage of complementary peptide cleavages by combining different fragmentation methods is the increased amount of information that is obtained during MS/MS analysis of modified peptides. On the basis of the obtained results, we are planning to design LC time-scale compatible, data-dependent MS/MS methods using the different fragmentation techniques in order to improve the identification and characterization of phosphopeptides.

Cyclotrons↗

PubMed2006

Post-translational modifications of steroid receptors.

The multiple physiological functions of steroid hormones have been known for many years. The cloning of the steroid receptors in the mid-1980s led to the concept of ligand-activated transcription factors and to the identification of specific DNA response elements in the regulatory regions of target genes. The next main development was the identification of cofactors with activating or repressing functions, of which several act by modifying histones and locally affecting the chromatin structure. Work from several groups shows that the steroid receptors themselves can also be modified at various positions. Besides the long-known phosphorylation at tyrosines and serine/threonine residues, other covalent additions such as acetylation, ubiquitylation and sumoylation have been evidenced for steroid receptors in recent years. These modifications affect receptor stability and activity, and provide potential mechanisms for cell- or gene-specific regulation. A better understanding of the impact of these post-translational modifications (PTMs) on steroid receptor function should help in the identification of novel ligands with improved clinical profiles.

Acetylation↗

PubMed2006

Immunohistochemical analysis of histone H3 acetylation and methylation--evidence for altered epigenetic signaling following traumatic brain injury in immature rats.

Posttranslational modifications (PTMs) of histone proteins may result in altered epigenetic signaling after pediatric traumatic brain injury (TBI). Hippocampal histone H3 acetylation and methylation in immature rats after moderate TBI were measured and decreased only in CA3 at 6 h and 24 h with persistent methylation decreases up to 72 h after injury. Decreased histone H3 acetylation and methylation suggest altered hippocampal CA3 epigenetic signaling during the first hours to days after TBI.

Acetylation↗

PubMed2025

A method for the detection and enrichment of endogenous cereblon substrates.

C-terminal cyclic imides are posttranslational modifications (PTMs) on proteins that are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN). Despite the observation of these modifications across the proteome by mass spectrometry-based proteomics, an orthogonal and generalizable method to visualize the C-terminal cyclic imide would enhance detection, sensitivity, and throughput of endogenous CRBN substrate characterization. Here, we develop an antibody-like reagent, termed "cerebody," for visualizing and enriching C-terminal cyclic imide-modified proteins. We describe the engineering of CRBN derivatives to produce cerebody and use it to identify CRBN substrates by western blot and enrichment from whole-cell and tissue lysates. CRBN substrates identified by cerebody enrichment are mapped, validated, and further characterized for dependence on the C-terminal cyclic imide modification. These methods will accelerate the characterization of endogenous CRBN substrates and their regulation.

Humans↗

PubMed2005

Synthesis of bifunctionalized mesoporous organosilica spheres for high-performance liquid chromatography.

Phenyl-functionalized mesoporous ethane-silicas with spherical morphology were synthesized by one-step co-condensation of phenyltrimethoxysilane (PTMS) and 1,2-bis(trimethoxysily)ethane (BTME) using a triblock copolymer EO(20)PO(70)EO(20) (P123) as template with the aid of a co-surfactant (cetyltrimethylammonium bromide, CTAB) and a co-solvent (ethanol) in acidic medium. Powder X-ray diffraction (XRD), nitrogen sorption measurement and scanning electron microscopy (SEM) show that phenyl-functionalized ethane-silica has wormhole-like mesostructure and perfect spherical morphology. The chemical stability of phenyl-functionalized mesoporous ethane-silica in basic medium is greatly improved owing to the ethane groups bridged in the mesoporous framework. This work also demonstrates that the phenyl-functionalized mesoporous ethane-silica spheres are excellent packing materials for potential application in the reversed-phase high-performance liquid chromatography (HPLC).

Chromatography, High Pressure Liquid↗

PubMed2004

Post-translational modifications of self antigens: implications for autoimmunity.

Alterations in amino acid sequence can generate neo-epitopes from self proteins, causing autoaggressive immune attack. There is a range of possible post-translational modifications (PTMs) of mammalian proteins that can allow immune recognition of neo-self epitopes. These effects can vary from overt increase in affinity of MHC or T-cell receptor binding, to more subtle effects on the activity of proteolytic enzymes involved in antigen processing. Furthermore, intriguing insights into how the complex interactions between inflammation, enzyme activity and protein modification can direct self recognition are beginning to be unearthed.

Animals↗

PubMed2004

iProLINK: an integrated protein resource for literature mining.

The exponential growth of large-scale molecular sequence data and of the PubMed scientific literature has prompted active research in biological literature mining and information extraction to facilitate genome/proteome annotation and improve the quality of biological databases. Motivated by the promise of text mining methodologies, but at the same time, the lack of adequate curated data for training and benchmarking, the Protein Information Resource (PIR) has developed a resource for protein literature mining--iProLINK (integrated Protein Literature INformation and Knowledge). As PIR focuses its effort on the curation of the UniProt protein sequence database, the goal of iProLINK is to provide curated data sources that can be utilized for text mining research in the areas of bibliography mapping, annotation extraction, protein named entity recognition, and protein ontology development. The data sources for bibliography mapping and annotation extraction include mapped citations (PubMed ID to protein entry and feature line mapping) and annotation-tagged literature corpora. The latter includes several hundred abstracts and full-text articles tagged with experimentally validated post-translational modifications (PTMs) annotated in the PIR protein sequence database. The data sources for entity recognition and ontology development include a protein name dictionary, word token dictionaries, protein name-tagged literature corpora along with tagging guidelines, as well as a protein ontology based on PIRSF protein family names. iProLINK is freely accessible at http://pir.georgetown.edu/iprolink, with hypertext links for all downloadable files.

Computational Biology↗

PubMed2005

Deep pain thresholds in the distal limbs of healthy human subjects.

Pressure pain thresholds (PPTs) in distal limbs have been under-investigated despite their potential clinical importance. Therefore, we compared PPTs over nail bed, bony prominences, and muscle in distal parts of upper and lower limbs. We investigated 12 healthy subjects using three handheld devices: a spring-loaded, analogue pressure threshold meter (PTM) with two operating ranges, and an electronic Algometer. PPTs were determined with three series of ascending stimulus intensities with a ramp of about 50 kPa/s. PPTs were normally distributed in logarithmic space. PPTs over different tissues varied significantly (ANOVA, p<0.001): mean thresholds and 95% confidence intervals were 615 kPa (266-1424 kPa) over the nail bed, 581 kPa (271-1245 kPa) over bony prominences, and 520 kPa (246-1100 kPa) over muscles. PPTs on the foot were higher than on the hand (ANOVA, p<0.01), except over muscles. PPTs were significantly lower with the Algometer than with PTMs (ANOVA, p<0.01); again these differences were least when testing over muscle. There was no significant right-left difference (ANOVA, p=0.33). In spite of considerable variability across subjects, reproducibility within subjects was high (correlation coefficients>0.90). For within-subject comparisons, threshold elevations beyond 33-43% would be abnormal (95% confidence intervals), whereas only deviations from the group mean by at least a factor of two would be abnormal with respect to absolute normative values. PPTs over distal muscles were comparable to published values on proximal limb and trunk muscles. These findings suggest that pressure pain testing over distal muscles may be a sensitive test for deep pain sensitivity and that the simple and less expensive devices are sufficient for testing this tissue type. Intra-individual site-to-site comparisons will be more sensitive than absolute normative values.

Adult↗

PubMed2004

Peptidomics.

Peptides occur in the whole animal kingdom, from the least evolved phyla with a very simple nervous system (coelenterates) to the highest vertebrates and are involved in most, if not all, physiological processes in animals. Knowing the amino acid sequence of peptide hormones or neurotransmitters is important since this allows for synthesis of large quantities of peptides to perform further functional analysis. Immunocytochemistry, radioimmunoassays (RIA), enzyme-linked immunosorbant assays (ELISA) and mass spectrometry can then provide information on the temporal and spatial distribution and quantification of the (neuro)peptide. Ever since the 1970s, a wealth of peptides has been discovered and investigated and this flow seems to be far from over. This is partially due to the use of new approaches mainly based on chromatographical purifications as well as molecular biological techniques. Surprisingly, peptides have so far been neglected in most proteomic studies. The finalization of the genome projects has opened new opportunities for rapid identification and functional analysis of (neuro)peptides as well. In analogy with the proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications (PTMs). This technology provides us with a fast and efficient tool to analyze the peptides from any tissue. This paper reviews the approaches that have been used so far to achieve this.

Amino Acid Sequence↗