The 4G10, pY20 and p-TYR-100 antibody specificity: profiling by peptide microarrays

Tinti, Michele; Nardozza, Aurelio Pio; Ferrari, Emanuela; Sacco, Francesca; Corallino, Salvatore; Castagnoli, Luisa

doi:10.1016/j.nbt.2011.12.001

Cited by 52 publications

(71 citation statements)

References 21 publications

(20 reference statements)

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“…These 11-mer peptides were chosen to compare the three EPIYA-motifs because α-phosphotyrosine antibodies typically recognize short phosphopeptides [35], [50], [55], [56]. Commonly, 11-mer peptides are also used for immunizations to generate phospho-specific antibodies, which then recognize the corresponding phosphopeptides bound to affinity columns and in ELISA (Biogenes, Berlin, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…These antibodies had been selected years ago to specifically detect a broad range of phosphorylated tyrosine residues in mammalian proteins. Three of these phosphotyrosine-specific antibodies have been shown to exhibit a similar phosphotyrosine-binding preference in mammalian proteins, preferably with a proline residue at position +3 and a leucine at position -1 [50]. However, proline and leucine residues are not present at this position in the corresponding phosphorylation sites of CagA [2], [5], [6].…”

Section: Introductionmentioning

confidence: 99%

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Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains

et al. 2014

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The clinical outcome of Helicobacter pylori infections is determined by multiple host-pathogen interactions that may develop to chronic gastritis, and sometimes peptic ulcers or gastric cancer. Highly virulent strains encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-sequence motifs, called A, B and C in Western-type strains, by members of the oncogenic Src and Abl host kinases. Phosphorylated EPIYA-motifs mediate interactions of CagA with host signaling factors – in particular various SH2-domain containing human proteins – thereby hijacking multiple downstream signaling cascades. Observations of tyrosine-phosphorylated CagA are mainly based on the use of commercial phosphotyrosine antibodies, which originally were selected to detect phosphotyrosines in mammalian proteins. Systematic studies of phosphorylated EPIYA-motif detection by the different antibodies would be very useful, but are not yet available. To address this issue, we synthesized phospho- and non-phosphopeptides representing each predominant Western CagA EPIYA-motif, and determined the recognition patterns of seven different phosphotyrosine antibodies in Western blots, and also performed infection studies with diverse representative Western H. pylori strains. Our results show that a total of 9–11 amino acids containing the phosphorylated EPIYA-motifs are necessary and sufficient for specific detection by these antibodies, but revealed great variability in sequence recognition. Three of the antibodies recognized phosphorylated EPIYA-motifs A, B and C similarly well; whereas preferential binding to phosphorylated motif A and motifs A and C was found with two and one antibodies, respectively, and the seventh anti-phosphotyrosine antibody did not recognize any phosphorylated EPIYA-motif. Controls showed that none of the antibodies recognized the corresponding non-phospho CagA peptides, and that all of them recognized phosphotyrosines in mammalian proteins. These data are valuable in judicious application of commercial anti-phosphotyrosine antibodies and in characterization of CagA phosphorylation during infection and disease development.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains

et al. 2014

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“…However, the three species (with the lowest molecular mass) seemed to be detected only by the γ-pY 47 antibody. Peptide microarray data has demonstrated that all phosphotyrosine antibody clones (i.e., 4G10, PY20, p-TYR-100) recognize their target in a sequence-specific context and that this recognition differs for each clone [21]. Our results demonstrate that this novel antibody, γ-pY 47 , recognizes its target sequence in the FcεRIγ with high selectivity and sensitivity, and recognizes more of the multiple molecular mass species (or modifications) of the FcεRIγthan the general anti-phosphotyrosine antibody 4G10.…”

Section: Generation Of a Novel Monoclonal Antibody That Specifically mentioning

confidence: 99%

Characterization of a Phospho-Specific Antibody to the Fcε Receptor γ Chain, Reveals Differences in the Regulation of Syk and Akt Phosphorylation

et al. 2013

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We previously demonstrated that the Fc receptor γ-chain Y 58 (C-terminal tyrosine) is highly susceptible to dephosphorylation; a mechanism that controls the extent of Syk activation and the downstream signaling in mast cells. Here, we explored the importance of the γ-chain Y 47 (N-terminal tyrosine) in mast cell signaling. We generated a highly sensitive and versatile phospho-specific antibody that recognized the phosphorylated Y 47 in various species. Using this antibody, we found that mutation of the FcεRIβ Y 219 to phenylalanine caused a loss in the phosphorylation of the γ-chain Y 47 , consistent with the previously described role of Y 219 in Lyn association with FcεRIβ and subsequent FcεRIγ phosphorylation. These conditions also diminished the tyrosine phosphorylation of Syk and LAT1 but, surprisingly, not the phosphorylation of Akt at T 308 . Mutation of Y 47 or Y 58 of the γ-chain also caused a marked inhibition of Syk and LAT1 phosphorylation, but only the latter mutant showed a reduction in Akt phosphorylation. These findings show that the full phosphorylation of Syk and LAT1 requires the FcεRIβ Y 219 and both Y 47 and Y 58 of the γ-chain. However, T 308 phosphorylation of Akt is largely independent of FcεRIγ Y 47 phosphorylation and of the Lyn-binding site (Y 219 ) on the FcεRIβ.

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“…Because of the level of phosphorylation and its role in many cell signaling processes, analytical techniques to enrich for protein phosphorylation have been developed. For example, antibody-based assays have been developed to study tyrosine phosphorylation (14,20,21), and metal affinity-based methods have been used to enrich pS, pT, and pY-containing peptides from proteolytic digests of cells and tissues (22,23). In combination with highly sensitive mass spectrometry workflows, these enrichment techniques have facilitated global phosphoproteomic studies in many biological systems (24 -27).…”

mentioning

confidence: 99%

Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes

Abelin

Patel

Lü

et al. 2016

Molecular & Cellular Proteomics

110

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Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ϳ95% of the reduced-representation phosphopeptide probes to be detected in ϳ200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.

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The 4G10, pY20 and p-TYR-100 antibody specificity: profiling by peptide microarrays

Cited by 52 publications

References 21 publications

Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains

Systematic Analysis of Phosphotyrosine Antibodies Recognizing Single Phosphorylated EPIYA-Motifs in CagA of Western-Type Helicobacter pylori Strains

Characterization of a Phospho-Specific Antibody to the Fcε Receptor γ Chain, Reveals Differences in the Regulation of Syk and Akt Phosphorylation

Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes

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