Characterization of SH2−Ligand Interactions via LibraryAffinity Selection with Mass Spectrometric Detection
Abstract
Synthetic combinatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening. Their use has greatly facilitated the study of protein-protein interactions. We have developed a practical technique for screening such libraries by integrating affinity chromatography selection with electrospray ionization mass spectrometric detection, referred to as library affinity selection-mass spectrometry (LAS-MS). The process allows for rapid and efficient screening of solution phase libraries and provides detailed information such as the relative affinities of substrates. The method is generally applicable to include nonpeptide libraries; moreover, electrospray tandem mass spectrometry (ES-MS/MS) yields sequence-specific identification of individual components without the need for chemical tags. This technique is demonstrated using the Src homology 2 (SH2) domain of phosphatidylinositol 3-kinase (PI 3-kinase). The critical importance of methionine in the position +3 (relative to the phosphotyrosine position) is demonstrated in a library built with a phosphotyrosine mimic, (phosphonodifluoromethyl)phenylalanine. The described method has broad applicability to combinatorial library screening.
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Native MS detects ligands directly bound to a protein, whereas affinity MS approaches typically measure ligand binding indirectly as free compounds.
“An important difference between native MS and affinity MS is that native MS detects ligands directly bound to a protein, whereas affinity MS approaches typically measure ligand binding indirectly as free compounds.”
From Native metabolomics identifies the rivulariapeptolide family of protease inhibitors (2022) · cites this paper as [24]
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- Authors
- Kelly, M. A., Liang, H., Sytwu, I.-I., Vlattas, I., Lyons, N. L., Bowen, B. R., & Wennogle, L. P.
- Affiliations
- CIBA Pharmaceuticals Division, 556 Morris Avenue, Summit, New Jersey 07901
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1996-01-01
- Volume
- 35
- Issue
- 36
- Pages
- 11747-11755
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W1994503397
- Cited by
- 63
- References
- 7
- Citations by year
- 2026: 1 · 2022: 2 · 2021: 1
- FWCI
- 5.11
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Keywords
- SH2 domain, phosphatidylinositol 3-kinase, combinatorial peptide libraries, protein-protein interaction, affinity chromatography, electrospray ionization tandem mass spectrometry
- MeSH terms
- Amino Acid Sequence, Chromatography, Affinity, Escherichia coli, Ligands, Methionine, Molecular Sequence Data, Peptides, Protein Binding, Recombinant Fusion Proteins, Mass Spectrometry, Gene Library, Phosphotransferases (Alcohol Group Acceptor), src Homology Domains, Phosphotyrosine, Phosphatidylinositol 3-Kinases
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