Biomedical subjects

Yehia Mechref

Publications and source records attributed to Yehia Mechref.

At least 19 recordsLinked to original sources

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↗

PubMed2007

Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

Carbohydrate Conformation↗

PubMed2007

Improved collision-induced dissociation analysis of peptides by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry through 3-sulfobenzoic acid succinimidyl ester labeling.

The sulfonation reagent, a succinimidyl ester of 3-sulfobenzoic acid, has been synthesized for effective peptide sequencing. It is capable of incorporating an additional mobile proton into the peptide backbone, thus, facilitating efficient collision-induced dissociation. This reagent is easily and inexpensively prepared in short time. Tandem mass spectra of the guanidinated and reagent-sulfonated peptides consist mainly of the y-ion series with higher intensities than those observed for solely guanidinated peptides. These enhanced tandem MS attributes significantly improved MASCOT total-ion scores, thus, allowing more confident peptide sequencing. This derivatization was also very effective for the analysis of tryptic digest of human blood serum proteins separated by two-dimensional gel electrophoresis. When used in LC-MALDI/MS/MS format, this type of derivatization does not adversely affect chromatographic efficiencies.

Amino Acid Sequence↗

PubMed2006

A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker.

Two isoforms of proliferating cell nuclear antigen (PCNA) have been observed in breast cancer cells. Commercially available antibodies to PCNA recognize both isoforms and, therefore, cannot differentiate between the PCNA isoforms in malignant and nonmalignant breast epithelial cells and tissues. We have developed a unique antibody that specifically detects a PCNA isoform (caPCNA) associated with breast cancer epithelial cells grown in culture and breast-tumor tissues. Immunostaining studies using this antibody suggest that the caPCNA isoform may be useful as a marker of breast cancer and that the caPCNA-specific antibody could potentially serve as a highly effective detector of malignancy. We also report here that the caPCNA isoform functions in breast cancer-cell DNA replication and interacts with DNA polymerase delta. Our studies indicate that the caPCNA isoform may be a previously uncharacterized detector of breast cancer.

Antibodies↗

PubMed2006

High-sensitivity profiling of glycoproteins from human blood serum through multiple-lectin affinity chromatography and liquid chromatography/tandem mass spectrometry.

We report here the use of high-performance lectin affinity enrichment of glycoproteins at microscale levels using a series of silica-bound lectins. The potential of this approach is being demonstrated for the glycoprotein enrichment from microliter volumes of human blood serum. Individual injections of sample to the affinity microcolumns packed with four lectin materials with different glycan specificities (Con A, SNA-I, UEA-I, PHA-L), followed by off-line reversed-phase pre-fractionation and nano-LC/MS/MS, permitted identification of 108 proteins in the lectin-bound fractions spanning a concentration dynamic range of 7-10 orders of magnitude. In contrast, multi-lectin microcolumn affinity chromatography, an alternative enrichment approach allowed identification of only 67 proteins. An attractive feature of high-performance lectin affinity chromatography at microscale levels is the substantial reduction of sample losses that are commonly experienced with extensive sample preparation needed for larger sample volumes.

Chromatography, Affinity↗

PubMed2006

Miniaturized separation techniques in glycomic investigations.

High-sensitivity glycomic analyses are becoming of a great interest in modern biomedical and clinical research, as well as in the development of recombinant protein products. The evolution of separation techniques for glycomic analysis at high sensitivity is highlighted in this review. These methodologies include capillary liquid chromatography, capillary electrophoresis (CE) and capillary electrochromatography (CEC). The potential of such methodologies in glycomic analysis is demonstrated for model glycoproteins as well as total glycomes derived from biological samples.

Chromatography, Liquid↗

PubMed2006

Determination of salsolinol and related catecholamines through on-line preconcentration and liquid chromatography/atmospheric pressure photoionization mass spectrometry.

A new analytical approach has been developed for simultaneous measurements of endogenous salsolinol and major catecholamines in brain tissue of experimental animals. This procedure involves a combination of on-line phenyl boronate affinity preconcentration and microcolumn liquid chromatography, followed by mass spectrometry equipped with an atmospheric pressure photoionization (APPI) source. Flow conditions of the APPI source were optimized for detection sensitivity while different dopants were evaluated. The on-line preconcentration was found essential for the sensitivity requirements of salsolinol measurements in the brain tissue from alcohol-preferring rats subjected to different levels of alcohol exposure.

Animals↗

PubMed2006

Changes in liver protein abundance in inbred alcohol-preferring rats due to chronic alcohol exposure, as measured through a proteomics approach.

This study compares the total liver proteome of inbred alcohol-preferring line (iP) rats exposed to alcohol with iP rats without alcohol experience. Rat liver proteins were extracted using a three-step procedure. Each of the three solutions solubilizes a different set of proteins. The extracted proteins were separated by 2-DE. Scanned gels of two sample groups, alcohol-exposed iP and alcohol-naïve iP, were compared, revealing many protein spots with significantly higher or lower densities. These spots were cut from the gel, destained, and subjected to trypsin digestion and subsequent identification by LC-MS/MS. Twenty-four individual rats, 12 alcohol-naïve, and 12 alcohol-exposed, were used in this study. Two groups, each containing six naïve and six exposed animals, were created for statistical comparison. For the first group, 64 spots were observed to have statistically significant intensity differences upon alcohol exposure across all three extracts while 118 such spots were found in the second group. There were 113 unique proteins in both groups together. The majority of these proteins were enzymes. Significant changes are observed for three major metabolic pathways: glycolysis, gluconeogenesis, and fatty acid beta-oxidation. In addition, enzymes involved in protein synthesis and antioxidant activity show significant changes in abundance in response to alcohol exposure.

Alcoholism↗

PubMed2006

Differentiating structural isomers of sialylated glycans by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry.

Using model acidic glycans, we demonstrate the benefits of permethylation for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) tandem mass spectrometry. With both the linear and branched structures, extensive cross-ring fragmentation product ions were generated, yielding valuable information on sugar linkages. Elimination of the negative charges commonly associated with sialylated structures through permethylation allowed their structural analysis in the positive ion mode. Extensive A- and X-type ions were observed for the linear structures, and slightly weaker signals for the branched sialylated structures. The diagnostic cross-ring fragments, permitting a distinction between alpha2-3 and alpha2-6 linkages of the sialic acid residues, were seen in abundance. Importantly, the cross-ring fragmentation with the branched structures provides adequate information to assign sialic acid residues, with a specific linkage, to a particular antenna.

Glycosides↗

PubMed2006

Semiautomated high-sensitivity profiling of human blood serum glycoproteins through lectin preconcentration and multidimensional chromatography/tandem mass spectrometry.

We describe an effective analytical approach to identify trace glycoproteins in a small volume of human serum. The system is based on automatable affinity enrichment through silica-based lectin microcolumns and a further separation of the retained glycoproteins on a reversed-phase liquid chromatography with superficially porous packing, operating at high temperature. The fractionated sample is further directed into a 96-well plate for trypsinization and LC-MS/MS analysis. Using a major-component depleted blood serum (16 microg total protein), we were able to identify 271 glycoproteins through this analytical system.

Chromatography, Affinity↗

PubMed2005

Combining lectin microcolumns with high-resolution separation techniques for enrichment of glycoproteins and glycopeptides.

Silica-based lectin microcolumns are described in this study together with the chemical procedures necessary for their preparation. The analytical merits of Canavalia ensiformis and Sambucus nigra lectins, [immobilized on activated macroporous silica], such as binding capacity, trapping reproducibility, and substrate selectivity, have been evaluated using model glycoproteins. The described microcolumns are applicable to high-pressure analytical schemes utilizing microvalving procedures, washing steps, and quantitative desorption for LC/MS analysis. The described analytical systems are amenable to the applications aiming at fractionation of complex glycopeptide mixtures and determination of the sites of glycosylation.

Amino Acid Sequence↗

PubMed2005

Modulation of differentiation-related gene 1 expression by cell cycle blocker mimosine, revealed by proteomic analysis.

L-mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G1 phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.

Amino Acid Sequence↗

PubMed2005

Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach.

Highly efficient separation techniques, laser-induced fluorescence (LIF) detection, and different mass-spectrometric (MS) measurements were combined in a multimethodological scheme to perform a comprehensive structural characterization of N-linked oligosaccharides in a murine monoclonal antibody (immunoglobulin G (IgG(kappa))). Monosaccharide compositional analysis was carried out through a capillary electrophoresis (CE)-LIF method, in which the chemically and enzymatically released sugars were fluorescently labeled. This analysis provides a preliminary assessment of certain structures, being followed by CE-LIF and matrix-assisted laser desorption/ionization (MALDI)-MS profiling of the intact glycan structures. Linkages and monosaccharide residues were confirmed by MALDI-MS in conjunction with exoglycosidase digestion. MALDI-MS and CE data were effectively combined to reveal the overall structural diversity of both acidic and neutral glycans. Finally, the sites of glycosylation and site occupancies were deduced through the measurements performed with microcolumn liquid chromatography coupled via electrospray to a quadrupole/time-of-flight instrument.

Amino Acid Sequence↗

PubMed2005

New hyphenated methodologies in high-sensitivity glycoprotein analysis.

High-sensitivity glycoprotein analyses are of particular interest in modern biomedical and clinical research, as well as in the development of recombinant protein products. The evolution of new hyphenated methodologies in high-sensitivity glycoprotein analysis is highlighted in this thematic review. These methodologies include, in particular, capillary LC/MALDI/TOF/TOF MS in conjunction with online permethylation platform, and silica-based lectin microcolumns interfaced to MS. The potential of these methodologies in glycomic and glycoproteomic analysis is demonstrated for model glycoproteins as well as total glycomes and glycoproteomes derived from biological samples. Additionally, the applications of CE-MS, CEC, and nanoLC with graphitized carbon in the areas of glycomics and glycoproteomics are described.

Chromatography, Ion Exchange↗

PubMed2005

Solid-phase permethylation of glycans for mass spectrometric analysis.

A miniaturized approach was developed for quantitative permethylation of oligosaccharides, which involves packing of sodium hydroxide powder in microspin columns or fused-silica capillaries (500 microm i.d.), permitting effective derivatization in less than a minute at microscale. Prior to mass spectrometry, analytes are mixed with methyl iodide in dimethyl sulfoxide solution containing traces of water before infusing through the microreactors. This procedure minimizes oxidative degradation and peeling reactions and avoids the need of excessive clean-up. Picomole amounts of linear and branched, sialylated and neutral glycan samples were rapidly and efficiently permethylated by this approach and analyzed by matrix-assisted laser desorption/ionization mass spectrometry.

Animals↗

PubMed2005

Automated interpretation of MS/MS spectra of oligosaccharides.

MOTIVATION: The emerging glycomics and glycoproteomics projects aim to characterize all forms of glycoproteins in different tissues and organisms. Tandem mass spectrometry (MS/MS) is the key experimental methodology for high-throughput glycan identification and characterization. Fragmentation of glycans from high energy collision-induced dissociation generates ions from glycosidic as well as internal cleavages. The cross-ring ions resulting from internal cleavages provide additional information that is important to reveal the type of linkage between monosaccharides. This information, however, is not incorporated into the current programs for analyzing glycan mass spectra. As a result, they can rarely distinguish from the mass spectra isomeric oligosaccharides, which have the same saccharide composition but different types of sequences, branches or linkages. RESULTS: In this paper, we describe a novel algorithm for glycan characterization using MS/MS. This algorithm consists of three steps. First, we develop a scoring scheme to identify potential bond linkages between monosaccharides, based on the appearance pattern of cross-ring ions. Next, we use a dynamic programming algorithm to determine the most probable oligosaccharide structures from the mass spectrum. Finally, we re-evaluate these oligosaccharide structures, taking into account the double fragmentation ions. We also show the preliminary results of testing our algorithm on several MS/MS spectra of oligosaccharides. AVAILABILITY: The program GLYCH is available upon request from the authors.

Algorithms↗

PubMed2004

Microdeposition device interfacing capillary electrochromatography and microcolumn liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry.

A sample deposition device has been constructed and optimized for interfacing CEC and capillary LC columns to MALDI mass spectrometry. For CEC analysis, the device is composed of an inlet buffer reservoir and an outlet buffer reservoir connected to a matrix reservoir through a connection sleeve. The matrix reservoir is connected to a deposition capillary via another connection sleeve. CEC eluent is transported to the matrix reservoir via a capillary that is connected to the deposition capillary by the connection sleeve inside the matrix reservoir. This connection sleeve also acts as a mixing chamber, allowing the CEC eluent to be mixed with matrix prior to deposition. Complex glycan mixtures can be separated by CEC using hydrophilic-phase monolithic columns, with capillary eluent being deposited on a standard MALDI plate along with a suitable matrix solution. Thousands of discrete, highly homogeneous dots can be generated for a subsequent mass spectrometric analysis. With minor modifications, this device is also applicable to capillary LC of peptides using gradient elution. In this configuration, the outlet of the LC column is connected to a deposition capillary inside a matrix reservoir through a connection sleeve that allows mixing of the LC effluent with an appropriate matrix. The device has been evaluated with the tryptic digests of proteins.

Chromatography, Liquid↗

PubMed2004

Enhanced post-source decay and cross-ring fragmentation of oligosaccharides facilitated by conversion to amino derivatives.

Post-source decay (PSD) fragmentation of chemically or enzymatically produced aminoglycans has been evaluated through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Conversion of native glycans to their respective aminoglycan derivatives improved detection sensitivity of the usual fragments and promoted cross-ring fragmentation of linear oligosaccharides, facilitating linkage recognition. The cross-ring fragmentations for both dextrin and dextran oligosaccharides were not limited to the reducing-end glucose moiety, as they were extended throughout the entire molecule. When the amino group was generated for N-glycans derived from three different glycoproteins, an enhancement of PSD was observed, without a significant extent of cross-ring fragmentation.

Amino Sugars↗