Evaluating the coupling efficiency of phosphorylated amino acids for SPOT synthesis

Tapia, Victor; Aÿ, Bernhard; Triebus, Julia; Wolter, Eike; Boisguérin, Prisca; Volkmer, Rudolf

doi:10.1002/psc.1068

Cited by 13 publications

(14 citation statements)

References 31 publications

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“…The peptides were released as amides from the N-CAPE membrane by using our standard two-step cleavage protocol for peptides synthesized on cellulose-bound linkers as reported previously. [38,68] The peptides were each dissolved in acetonitrile/water (1:1, 100 mL).…”

Section: Discussionmentioning

confidence: 99%

“…The determined quality and purity of the SPOTsynthesized peptides are adequate for a reliable screening study. [34,38,39] Importance of peptide length Antibody epitopes usually do not exceed about 15 amino acids in length. However, by using synthetic peptides as antigenic probes, it has been shown that linear peptide epitopes are constructed by two to five critical residues, usually located within a sequence between five and eight amino acids in length.…”

Section: Quality Of Membrane-bound Peptidesmentioning

confidence: 99%

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Identification of IgE Binding to Api g 1‐Derived Peptides

et al. 2010

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Celery is a frequent cause of food allergy in pollen-sensitized patients and can induce severe allergic reactions. Clinical symptoms cannot be predicted by skin prick tests (SPTs) or by determining allergen-specific immunoglobulin E (IgE). Our aim was to identify specific IgE binding peptides by using an array technique. For our study, the sera of 21 patients with positive double-blind, placebo-controlled food challenge (DBPCFC) to celery, as well as the sera of 17 healthy patients were used. Additionally, all patients underwent skin tests along with determinations of specific IgE binding. The major allergen of celery Api g 1.0101 (Apium graveolens) was synthesized as an array of overlapping peptides and probed with the patients' sera. We developed an improved immunoassay protocol by investigating peptide lengths, peptide densities, incubation parameters, and readout systems, which could influence IgE binding. Sera of celery-allergic patients showed binding to three distinct regions of Api g 1.0101. The region including amino acids 100 to 126 of Api g 1.0101 is the most important region for IgE binding. This region caused a fivefold higher binding of IgE from the sera of celery-allergic patients compared to those of healthy individuals. In particular, one peptide (VLVPTADGGSIC) was recognized by all sera of celery-allergic patients. In contrast, no binding to this peptide was detected in sera of the healthy controls. Our improved assay strategy allows us to distinguish between celery-allergic and healthy individuals, but needs to be explored in a larger cohort of well-defined patients.

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Section: Discussionmentioning

confidence: 99%

Section: Quality Of Membrane-bound Peptidesmentioning

confidence: 99%

Identification of IgE Binding to Api g 1‐Derived Peptides

et al. 2010

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“…SPOT technology simplified the chemical synthesis of peptide arrays to the addressable deposition of reagents on a planar cellulose membrane (filter paper). Moreover, chemical synthesis allows one to incorporate phosphorylated [40], methylated or acetylated amino acids [41,42], use non-natural building blocks [43,44], prepare branched and cyclic structures [45] and label with chromophores or short biological tags such as biotin [46,47].…”

Section: Spot Synthesis and Synthetic Peptide Arraysmentioning

confidence: 99%

Synthetic peptide arrays for investigating protein interaction domains

2012

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Synthetic peptide array technology was first developed in the early 1990s by Ronald Frank. Since then the technique has become a powerful tool for high throughput approaches in biology and biochemistry. Here, we focus on peptide arrays applied to investigate the binding specificity of protein interaction domains such as WW, SH3, and PDZ domains. We describe array‐based methods used to reveal domain networks in yeast, and briefly review rules as well as ideas about the synthesis and application of peptide arrays. We also provide initial results of a study designed to investigate the nature and evolution of SH3 domain interaction networks in eukaryotes.

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“…SPOT synthesis technology (14) was used to synthesize a panel of 51 13-mer peptides corresponding to all the tyrosine residues present in SHP-2, in their nonphosphorylated and phosphorylated forms, on a cellulose membrane (43). Membranes were incubated with the SHP-2 N-SH2/C-SH2 and GRB2 SH2 domains expressed as GST fusion proteins.…”

Section: C459smentioning

confidence: 99%

Reactive Oxygen Species and Epidermal Growth Factor Are Antagonistic Cues Controlling SHP-2 Dimerization

Nardozza

D’Orazio

Trapannone

et al. 2012

Molecular and Cellular Biology

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The SHP-2 tyrosine phosphatase plays key regulatory roles in the modulation of the cell response to growth factors and cytokines. Over the past decade, the integration of genetic, biochemical, and structural data has helped in interpreting the pathological consequences of altered SHP-2 function. Using complementary approaches, we provide evidence here that endogenous SHP-2 can dimerize through the formation of disulfide bonds that may also involve the catalytic cysteine. We show that the fraction of dimeric SHP-2 is modulated by growth factor stimulation and by the cell redox state. Comparison of the phosphatase activities of the monomeric self-inhibited and dimeric forms indicated that the latter is 3-fold less active, thus pointing to the dimerization process as an additional mechanism for controlling SHP-2 activity. Remarkably, dimers formed by different SHP-2 mutants displaying diverse biochemical properties were found to respond differently to epidermal growth factor (EGF) stimulation. Although this differential behavior cannot be rationalized mechanistically yet, these findings suggest a possible regulatory role of dimerization in SHP-2 function. SHP-2 is a ubiquitously expressed nontransmembrane protein tyrosine phosphatase (PTP) modulating the cell response to growth factors and cytokines (29). Numerous lines of evidence indicate that this phosphatase promotes the activation of growth pathways and that mutations altering its activity cause congenital or somatic pathologies, such as Noonan and LEOPARD syndromes or leukemia (46, 47). SHP-2 has a simple domain structure in which the enzymatic PTP domain is flanked at the aminoterminal side by two SH2 domains and at the carboxy-terminal side by a relatively unstructured region of approximately 70 residues containing a notable proline-rich motif and a few tyrosine residues that were found to be phosphorylated in phosphoproteomic studies (18,49). Somewhat contradicting the popular model that phosphatases play a negative role in controlling hormone-dependent signal transduction, SHP-2 is one of the rare tyrosine phosphatases which reinforces activation signals rather than dampening them (37, 42, 52). In the absence of growth factors, SHP-2 has a low basal catalytic activity, whereas it is rapidly activated after growth factor addition to the culture medium (7,15). A combination of genetic and structural studies have permitted elaboration of an elegant molecular model to explain the pathological consequences of a large number of mutations that are predicted to affect the conformation of the phosphatase (19,24,45). The crystallographic structure of SHP-2 shows that the aminoterminal SH2 domain makes extensive contacts with the phosphatase substrate pocket, thus offering a molecular explanation for its low basal activity (15). This model is supported by the observation that the majority of activating mutations causing either the Noonan syndrome or leukemia weaken the interaction between the SH2 and phosphatase domains, thus causing exposure of the phosphatase acti...

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Evaluating the coupling efficiency of phosphorylated amino acids for SPOT synthesis

Cited by 13 publications

References 31 publications

Identification of IgE Binding to Api g 1‐Derived Peptides

Identification of IgE Binding to Api g 1‐Derived Peptides

Synthetic peptide arrays for investigating protein interaction domains

Reactive Oxygen Species and Epidermal Growth Factor Are Antagonistic Cues Controlling SHP-2 Dimerization

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