Engineering SH2 Domains with Tailored Specificities and Affinities

Martyn, Gregory D.; Veggiani, Gianluca; Sidhu, Sachdev S.

doi:10.1007/978-1-0716-3393-9_17

Cited by 2 publications

(2 citation statements)

References 45 publications

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“…Here, short chain protein domains have been constructed and affinity enhanced through yeast two hybrid screening to arrive at high affinity matrices capable of outperforming traditional IMAC approaches. 337,338 Tips for Studying Phosphorylation.…”

Section: Peptide Enrichmentmentioning

confidence: 99%

“…More recently, the use of Src Homology 2 (SH2) domains as specific affinity reagents for phosphotyrosine is an emerging technology allowing an expanded fractionation of tyrosine phosphopeptides. Here, short chain protein domains have been constructed and affinity enhanced through yeast two hybrid screening to arrive at high affinity matrices capable of outperforming traditional IMAC approaches. , …”

Section: Enrichment and Depletionmentioning

confidence: 99%

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Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

Jiang,

Rex,

Schuster

et al. 2024

ACS Meas. Sci. Au

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Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. “Shotgun proteomics” or “bottom-up proteomics” is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this Review will serve as a handbook for researchers who are new to the field of bottom-up proteomics.

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Section: Peptide Enrichmentmentioning

confidence: 99%

Section: Enrichment and Depletionmentioning

confidence: 99%

Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

Jiang,

Rex,

Schuster

et al. 2024

ACS Meas. Sci. Au

View full text Add to dashboard Cite

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Engineered FHA domains can bind to a variety of Phosphothreonine-containing peptides

Thota,

Allen,

Grahn

et al. 2024

Protein Engineering, Design and Selection

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Antibodies play a crucial role in monitoring post-translational modifications, like phosphorylation, which regulates protein activity and location; however, commercial polyclonal and monoclonal antibodies have limitations in renewability and engineering compared to recombinant affinity reagents. A scaffold based on the Forkhead-associated domain (FHA) has potential as a selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), with limited cross-reactivity were isolated from an M13 bacteriophage display library by affinity selection with phosphopeptides corresponding to human mTOR, Chk2, 53BP1, and Akt1 proteins. To determine the specificity of the representative pTBDs, we focused on binders to the pT543 phosphopeptide (536-IDEDGENpTQIEDTEP-551) of the DNA repair protein 53BP1. ELISA and western blot experiments have demonstrated the pTBDs are specific to phosphothreonine, demonstrating the potential utility of pTBDs for monitoring the phosphorylation of specific threonine residues in clinically relevant human proteins.

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Engineering SH2 Domains with Tailored Specificities and Affinities

Cited by 2 publications

References 45 publications

Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

Engineered FHA domains can bind to a variety of Phosphothreonine-containing peptides

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