SPOT synthesis: Reliability of array-based measurement of peptide binding affinity

Weiser, Armin A.; Or-Guil, Michal; Tapia, Victor; Leichsenring, Astrid; Schuchhardt, Johannes; Frömmel, Cornelius; Volkmer, Rudolf

doi:10.1016/j.ab.2005.04.033

Cited by 59 publications

(64 citation statements)

References 22 publications

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“…1D). Interactions identified in this study correlate with high affinity binding events measured quantitatively in solution suggesting that the results are semiquantitative (27,28). Of 21 array-positive interactions tested, all had K d values in the low micromolar or better (Fig.…”

Section: Sh2 Domains Are Highly Selective For Physiological Peptidementioning

confidence: 65%

“…The use of peptide arrays to analyze protein peptide interactions is well established and highly reproducible (28). "Array-positive" interactions were ascribed where the intensity of the signal exceeded the mean intensity of the spots on that membrane by 3-fold.…”

Section: Sh2 Domains Are Highly Selective For Physiological Peptidementioning

confidence: 99%

“…Peptide arrays using the SPOT method provide a semiquantitative approach to studying protein-protein interactions (23,25,27,28). Specificity profiling and analysis of protein domain interactions have been well captured and described using this approach (10,23,25).…”

mentioning

confidence: 99%

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SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

Liu¹,

Jablonowski²,

Shah³

et al. 2010

Molecular & Cellular Proteomics

106

137

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Selective ligand recognition by modular protein interaction domains is a primary determinant of specificity in signaling pathways. Src homology 2 (SH2) domains fulfill this capacity immediately downstream of tyrosine kinases, acting to recruit their host polypeptides to ligand proteins harboring phosphorylated tyrosine residues. The degree to which SH2 domains are selective and the mechanisms underlying selectivity are fundamental to understanding phosphotyrosine signaling networks. An examination of interactions between 50 SH2 domains and a set of 192 phosphotyrosine peptides corresponding to physiological motifs within FGF, insulin, and IGF-1 receptor pathways indicates that individual SH2 domains have distinct recognition properties and exhibit a remarkable degree of selectivity beyond that predicted by previously described binding motifs. The underlying basis for such selectivity is the ability of SH2 domains to recognize both permissive amino acid residues that enhance binding and non-permissive amino acid residues that oppose binding in the vicinity of the essential phosphotyrosine. Neighboring positions affect one another so local sequence context matters to SH2 domains. This complex linguistics allows SH2 domains to distinguish subtle differences in peptide ligands. This newly appreciated contextual dependence substantially increases the accessible information content embedded in the peptide ligands that can be effectively integrated to determine binding. This concept may serve more broadly as a paradigm for subtle recognition of physiological ligands by protein interaction domains. Molecular & Cellular Proteomics 9:2391-2404, 2010. The Src homology 2 (SH2)1 domain is the classic archetype for the large family of modular protein interaction domains that serve to organize a diverse array of cellular processes. As its name suggests, the SH2 domain was identified in the regulatory regions of non-receptor protein-tyrosine kinases (PTKs) of the Src family (1). The human genome encodes 110 SH2 domain proteins (2) that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. SH2 domains interact with phosphorylated tyrosinecontaining peptide sequences (3-6). In doing so, they couple activated PTKs to intracellular pathways that regulate many aspects of cellular communication in metazoans (7,8). In this manner, SH2 domains function alongside PTKs and proteintyrosine phosphatases to direct specificity in phosphotyrosine signaling. SH2 domain proteins play a critical role in development and have been linked to a wide range of human diseases including cancers, diabetes, and immunodeficiencies (2).Activation and recruitment of PTKs and protein-tyrosine phosphatases can control the spatial and temporal organization of phosphotyrosine signaling. However, signaling specificity in terms of downstream targets is entirely determined by phosphotyrosine-mediated recruitment events for which SH2 domains are responsible. Therefore, the binding selectivity of SH2 domains is critical for ...

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Section: Sh2 Domains Are Highly Selective For Physiological Peptidementioning

confidence: 65%

Section: Sh2 Domains Are Highly Selective For Physiological Peptidementioning

confidence: 99%

See 1 more Smart Citation

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

Liu¹,

Jablonowski²,

Shah³

et al. 2010

Molecular & Cellular Proteomics

106

137

View full text Add to dashboard Cite

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“…Visual inspection of the SPOT array results reveals that both the N-terminal (residues 1-10) and the C-terminal sequence regions (residues 24 -30) of MACFp1 tolerate any substitution. The signal intensities of individual spots (referred to as SI values) can be used as an objective criteria to assess the binding efficiency of peptide variants (38). We defined the SI threshold that discriminates between binding and non-binding as half of the mean value of the wild type SI.…”

Section: Sequence Profiling Of the Mtls Sequence From Macf2-tomentioning

confidence: 99%

Sequence Determinants of a Microtubule Tip Localization Signal (MtLS)

Buey

Sen

Kortt

et al. 2012

Journal of Biological Chemistry

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Background: Microtubule plus-end-tracking proteins (ϩTIPs) use microtubule tip localization signals (MtLSs) to target growing microtubule ends in an end-binding protein (EB)-dependent manner. Results: The data define the sequence determinants of a canonical MtLS. Conclusion: EB binding affinity and microtubule-tip tracking activity correlate. Significance: The data provide a basis to carry out genome-wide predictions of novel ϩTIPs.

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“…The limitations of reliability of SPOT technology have been described by several authors [18][19][20] . Since purity and quantity of the peptides synthesized on cellulose may vary, all results achieved using this method need to be confirmed with peptides produced by conventional solid-phase peptide synthesis on resin.…”

Section: Experimental Designmentioning

confidence: 99%

Use of luminescent bacteria for rapid screening and characterization of short cationic antimicrobial peptides synthesized on cellulose using peptide array technology

Hilpert

Hancock

2007

Nat Protoc

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The increasing multi-resistance of pathogenic bacteria requires the development of novel classes of antibiotics. Antimicrobial host defense peptides represent one promising class. Here we describe a protocol for screening large numbers of peptides against any microbe of interest. Peptides synthesized on a cellulose support by peptide array technology can be added to a microbe that expresses the luxCDABE (luciferase) gene cassette. Any substance that decreases the energy level within the microbe will cause a quantifiable decrease in light production. The potency of the compound, at different concentrations, is reflected by the rate of decrease in luminescence. In conjunction with peptide array technology, the screening assay is rapid and high throughput and demonstrates good correlation with conventional killing or minimal inhibitory concentration assays performed with the same peptides synthesized by standard solid-phase peptide synthesis. The protocol can be completed in 3 d.

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SPOT synthesis: Reliability of array-based measurement of peptide binding affinity

Cited by 59 publications

References 22 publications

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

Sequence Determinants of a Microtubule Tip Localization Signal (MtLS)

Use of luminescent bacteria for rapid screening and characterization of short cationic antimicrobial peptides synthesized on cellulose using peptide array technology

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