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Clinical Assay for the Early Detection of Colorectal Cancer Using Mass Spectrometric Wheat Germ Agglutinin Multiple Reaction Monitoring.

ABSTRACT: Colorectal cancer (CRC) is currently the third leading cause of cancer-related mortality in the world. U.S. Food and Drug Administration-approved circulating tumor markers, including carcinoembryonic antigen, carbohydrate antigen (CA) 19-9 and CA125 were used as prognostic biomarkers of CRC that attributed to low sensitivity in diagnosis of CRC. Therefore, our purpose is to develop a novel strategy for novel clinical biomarkers for early CRC diagnosis. We used mass spectrometry (MS) methods such as nanoLC-MS/MS, targeted LC-MS/MS, and stable isotope-labeled multiple reaction monitoring (MRM) MS coupled to test machine learning algorithms and logistic regression to analyze plasma samples from patients with early-stage CRC, late-stage CRC, and healthy controls (HCs). On the basis of our methods, 356 peptides were identified, 6 differential expressed peptides were verified, and finally three peptides corresponding wheat germ agglutinin (WGA)-captured proteins were semi-quantitated in 286 plasma samples (80 HCs and 206 CRCs). The novel peptide biomarkers combination of PF4^54-62, ITIH4^429-438, and APOE^198-207 achieved sensitivity 84.5%, specificity 97.5% and an AUC of 0.96 in CRC diagnosis. In conclusion, our study demonstrated that WGA-captured plasma PF4^54-62, ITIH4^429-438, and APOE^198-207 levels in combination may serve as highly effective early diagnostic biomarkers for patients with CRC.

SUBMITTER: Tsai IJ

PROVIDER: S-EPMC8124906 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Clinical Assay for the Early Detection of Colorectal Cancer Using Mass Spectrometric Wheat Germ Agglutinin Multiple Reaction Monitoring

Project description:Colorectal cancer (CRC) is currently the third leading cause of cancer related mortality in the world. U.S. Food and Drug Administration-approval circulating tumor markers, including carcinoembryonic antigen, carbohydrate antigen (CA) 19-9 and CA125, were used as prognostic biomarker of CRC that attributed to low sensitivity in diagnosis of CRC. Therefore, our purpose is to develop a novel strategy for novel clinical biomarker for early CRC diagnosis. We used mass spectrometry (MS) methods such as nanoLC-MS/MS, targeted LC-MS/MS, and stable iso-tope-labeled multiple reaction monitoring (MRM) MS coupled to machine learning algorithms and logistic regression to analyze plasma samples from patients with early-stage CRC, late-stage CRC, and healthy controls (HCs).

2021-07-29 | PXD024997 | Pride

High-affinity multivalent wheat germ agglutinin ligands by one-pot click reaction.

Project description:A series of six mono-, di-, and trivalent N,N'-diacetylchitobiose derivatives was conveniently prepared by employing a one-pot procedure for Cu(II)-catalyzed diazo transfer and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) starting from commercially available amines. These glycoclusters were probed for their binding potencies to the plant lectin wheat germ agglutinin (WGA) from Triticum vulgaris by an enzyme-linked lectin assay (ELLA) employing covalently immobilized N-acetylglucosamine (GlcNAc) as a reference ligand. IC(50) values were in the low micromolar/high nanomolar range, depending on the linker between the two disaccharides. Binding enhancements β up to 1000 for the divalent ligands and 2800 for a trivalent WGA ligand, compared to N,N'-diacetylchitobiose as the corresponding monovalent ligand, were observed. Molecular modeling studies, in which the chitobiose moieties were fitted into crystallographically determined binding sites of WGA, correlate the binding enhancements of the multivalent ligands with their ability to bind to the protein in a chelating mode. The best WGA ligand is a trivalent cluster with an IC(50) value of 220 nM. Calculated per mol of contained chitobiose, this is the best WGA ligand known so far.

| S-EPMC3388869 | biostudies-literature

Folding and homodimerization of wheat germ agglutinin.

Project description:Wheat germ agglutinin (WGA) is emblematic of proteins that specialize in the recognition of carbohydrates. It was the first lectin reported to have a capacity for discriminating between normal and malignant cells. Since then, it has become a preferred model for basic research and is frequently considered in the development of biomedical and biotechnological applications. However, the molecular basis for the structural stability of this homodimeric lectin remains largely unknown, a situation that limits the rational manipulation and modification of its function. In this work we performed a thermodynamic characterization of WGA folding and self-association processes as a function of pH and temperature by using differential scanning and isothermal dilution calorimetry. WGA is monomeric at pH 2, and one of its four hevein-like domains is unfolded at room temperature. Under such conditions, the agglutinin exhibits a fully reversible thermal unfolding that consists of three two-state transitions. At higher pH values, the protein forms weak, nonobligate dimers. This behavior contrasts with that observed for the other plant lectins studied thus far, which form strong, obligate oligomers, indicating a distinctly different molecular basis for WGA function. For dimer formation, the four domains must be properly folded. Nevertheless, depending on the solution conditions, self-association may be coupled with folding of the labile domain. Therefore, dimerization may proceed as a rigid-body-like association or a folding-by-binding event. This hybrid behavior is not seen in other plant lectins. The emerging molecular picture for the WGA assembly highlights the need for a reexamination of existing ligand-binding data in the literature.

| S-EPMC3177061 | biostudies-literature

Multiple-reaction monitoring-mass spectrometric assays can accurately measure the relative protein abundance in complex mixtures.

Project description:BACKGROUND:Mass spectrometric assays could potentially replace protein immunoassays in many applications. Previous studies have demonstrated the utility of liquid chromatography-multiple-reaction monitoring-mass spectrometry (LC-MRM/MS) for the quantification of proteins in biological samples, and many examples of the accuracy of these approaches to quantify supplemented analytes have been reported. However, a direct comparison of multiplexed assays that use LC-MRM/MS with established immunoassays to measure endogenous proteins has not been reported. METHODS:We purified HDL from the plasma of 30 human donors and used label-free shotgun proteomics approaches to analyze each sample. We then developed 2 different isotope-dilution LC-MRM/MS 6-plex assays (for apoliporoteins A-I, C-II, C-III, E, B, and J): 1 assay used stable isotope-labeled peptides and the other used stable isotope-labeled apolipoprotein A-I (an abundant HDL protein) as an internal standard to control for matrix effects and mass spectrometer performance. The shotgun and LC-MRM/MS assays were then compared with commercially available immunoassays for each of the 6 analytes. RESULTS:Relative quantification by shotgun proteomics approaches correlated poorly with the 6 protein immunoassays. In contrast, the isotope dilution LC-MRM/MS approaches showed correlations with immunoassays of r = 0.61-0.96. The LC-MRM/MS approaches had acceptable reproducibility (<13% CV) and linearity (r ?0.99). Strikingly, a single protein internal standard applied to all proteins performed as well as multiple protein-specific peptide internal standards. CONCLUSIONS:Because peak area ratios measured in multiplexed LC-MRM/MS assays correlate well with immunochemical measurements and have acceptable operating characteristics, we propose that LC-MRM/MS could be used to replace immunoassays in a variety of settings.

| S-EPMC3665768 | biostudies-literature

Homotypic endothelial nanotubes induced by wheat germ agglutinin and thrombin.

Project description:Endothelial barrier formation is maintained by intercellular communication through junctional proteins. The mechanisms involved in maintaining endothelial communication subsequent to barrier disruption remain unclear. It is known that low numbers of endothelial cells can be interconnected by homotypic actin-driven tunneling nanotubes (TNTs) which could be important for intercellular transfer of information in vascular physiology. Here we sought insight into the triggers for TNT formation. Wheat germ agglutinin, a C-type lectin and known label for TNTs, unexpectedly caused striking induction of TNTs. A succinylated derivative was by contrast inactive, suggesting mediation by a sialylated protein. Through siRNA-mediated knockdown we identified that this protein was likely to be CD31, an important sialylated membrane protein normally at endothelial cell junctions. We subsequently considered thrombin as a physiological inducer of endothelial TNTs because it reduces junctional contact. Thrombin reduced junctional contact, redistributed CD31 and induced TNTs, but its effect on TNTs was CD31-independent. Thrombin-induced TNTs nevertheless required PKCα, a known mediator of thrombin-dependent junctional remodelling, suggesting a necessity for junctional proteins in TNT formation. Indeed, TNT-inducing effects of wheat germ agglutinin and thrombin were both correlated with cortical actin rearrangement and similarly Ca2+-dependent, suggesting common underlying mechanisms. Once formed, Ca2+ signalling along TNTs was observed.

| S-EPMC5953990 | biostudies-literature

Subfractionation of cardiac sarcolemma with wheat-germ agglutinin.

Project description:The properties of highly purified bovine cardiac sarcolemma subfractionated with the lectin, wheat-germ agglutinin (WGA) were studied. Two different membrane subfractions were isolated, one which was agglutinated in the presence of 1.0 mg of WGA/mg of protein (WGA+ vesicles) and a second fraction which failed to agglutinate (WGA- vesicles). These two membrane fractions had quantitatively different rates of Na+/K+-dependent, ouabain-sensitive ATPase and Na+/Ca2+ exchange activities, yet a similar protein composition, which suggests that they were both derived from the plasma membrane. WGA- vesicles had a decreased number of [3H]quinuclidinyl benzilate-binding sites and no detectable [3H]nitrendipine-binding sites. Electron-microscopic and freeze-fracture analysis showed that the WGA+ fraction was composed of typical spherical sarcolemmal vesicles, whereas the WGA- fraction primarily contained elongated tubular structures suggestive of the T-tubule vesicles which were previously isolated from skeletal muscle. Assays of marker enzymes revealed that these fractions were neither sarcoplasmic reticulum nor plasma membrane from endothelial cells. Moreover, WGA agglutination did not result in the separation of right-side-out and inside-out vesicles. On the basis of these findings we propose that the WGA+ fraction corresponds to highly purified sarcolemma, whereas the WGA- fraction may be derived from T-tubule membranes.

| S-EPMC1133668 | biostudies-other

The purification and characterization of wheat-germ agglutinin.

Project description:The purification of wheat-germ agglutinin by precipitation with ammonium sulphate and by chromatography on Sephadex G-75, Sepharose-ovomucoid and CM-cellulose is described. This procedure gave agglutinin preparations which were homogeneous on polyacrylamide gels under a variety of conditions. Purified wheat-germ agglutinin formed colourless solutions and was relatively insoluble at neutral pH; maximum solubility in 1mm-tris-HCl buffer, pH7.4, was approx. 1mg/ml. The agglutinin was a glycoprotein containing a single polypeptide chain with an approximate molecular weight of 23000. The N-terminus of the oxidized agglutinin was cysteic acid and the C-terminus was glycine. The amino acid composition showed that the protein was extremely rich in cysteine and cystine; there were 15-17 free SH groups/mol. The absorption maximum for the protein was at 272nm and the molar extinction coefficient at 280nm was 1.09x10(5) litre.mol(-1).cm(-1). Equilibrium dialysis indicated that there was only one binding site per molecule for N-acetylglucosamine.

| S-EPMC1174230 | biostudies-other

The purification, composition and specificity of wheat-germ agglutinin.

Project description:1. The purification of wheat-germ agglutinin from commercial wheat germ is described. By ion-exchange chromatography three active proteins (isolectins) were separated, one of which was examined in detail. 2. The amino acid composition is unusual, as 20% of residues are half-cystine and 21% are glycine. Unlike most lectins and contrary to previous reports, this protein is not a glycoprotein. 3. The efficiency of various saccharides as inhibitors of the agglutination reaction was investigated and from this the specificity of the binding site was inferred. Of monosaccharides, only derivatives of glucose with a 2-acetamido group and a free 3-hydroxyl group are effective inhibitors, and glycosides of either anomeric configuration are bound. Oligosaccharides are much more powerful inhibitors of agglutination than are monosaccharides. 4. It is proposed that the binding site consists of three or four subsites with differing specificities, in a cleft in the molecule resembling that proposed for hen's-egg-white lysozyme.

| S-EPMC1177449 | biostudies-other

COPD Exacerbation Biomarkers Validated Using Multiple Reaction Monitoring Mass Spectrometry.

Project description:BackgroundAcute exacerbations of chronic obstructive pulmonary disease (AECOPD) result in considerable morbidity and mortality. However, there are no objective biomarkers to diagnose AECOPD.MethodsWe used multiple reaction monitoring mass spectrometry to quantify 129 distinct proteins in plasma samples from patients with COPD. This analytical approach was first performed in a biomarker cohort of patients hospitalized with AECOPD (Cohort A, n = 72). Proteins differentially expressed between AECOPD and convalescent states were chosen using a false discovery rate <0.01 and fold change >1.2. Protein selection and classifier building were performed using an elastic net logistic regression model. The performance of the biomarker panel was then tested in two independent AECOPD cohorts (Cohort B, n = 37, and Cohort C, n = 109) using leave-pair-out cross-validation methods.ResultsFive proteins were identified distinguishing AECOPD and convalescent states in Cohort A. Biomarker scores derived from this model were significantly higher during AECOPD than in the convalescent state in the discovery cohort (p<0.001). The receiver operating characteristic cross-validation area under the curve (CV-AUC) statistic was 0.73 in Cohort A, while in the replication cohorts the CV-AUC was 0.77 for Cohort B and 0.79 for Cohort C.ConclusionsA panel of five biomarkers shows promise in distinguishing AECOPD from convalescence and may provide the basis for a clinical blood test to diagnose AECOPD. Further validation in larger cohorts is necessary for future clinical translation.

| S-EPMC4985129 | biostudies-literature

A novel method for live imaging of human airway cilia using wheat germ agglutinin.

Project description:Multiciliated epithelial cells in the airway are essential for mucociliary clearance. Their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. Epithelial surfaces were observed using an epi-fluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.

| S-EPMC7468155 | biostudies-literature

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