biostudies-literatureBonham CANatural Sciences and Engineering Research Council of CanadaCold Spring Harbor LaboratoryNational Institutes of HealthShanghaiTech UniversityDon Monti Memorial Research Foundation104582https://www.ebi.ac.uk/biostudies/studies/S-EPMC10148153biostudies-literatureUnknown299(5)The ability to define functional interactions between enzymes and their substrates is crucial for understanding biological control mechanisms; however, such methods face challenges in the transient nature and low stoichiometry of enzyme-substrate interactions. Now, we have developed an optimized strategy that couples substrate-trapping mutagenesis to proximity-labeling mass spectrometry for quantitative analysis of protein complexes involving the protein tyrosine phosphatase PTP1B. This methodology represents a significant shift from classical schemes; it is capable of being performed at near-endogenous expression levels and increasing stoichiometry of target enrichment without a requirement for stimulation of supraphysiological tyrosine phosphorylation levels or maintenance of substrate cThe Journal of biological chemistryCoupling substrate-trapping with proximity-labeling to identify protein tyrosine phosphatase PTP1B signaling networks.PMC10148153CA53840CA45508Singh RKMandati VTonks NKPappin DJBonham CAfalseCoupling substrate-trapping with proximity-labeling to identify protein tyrosine phosphatase PTP1B signaling networks.The ability to define functional interactions between enzymes and their substrates is crucial for understanding biological control mechanisms; however, such methods face challenges in the transient nature and low stoichiometry of enzyme-substrate interactions. Now, we have developed an optimized strategy that couples substrate-trapping mutagenesis to proximity-labeling mass spectrometry for quantitative analysis of protein complexes involving the protein tyrosine phosphatase PTP1B. This methodology represents a significant shift from classical schemes; it is capable of being performed at near-endogenous expression levels and increasing stoichiometry of target enrichment without a requirement for stimulation of supraphysiological tyrosine phosphorylation levels or maintenance of substrate c2023-01-01T00:00:00Z2023 Mar2025-04-21T17:24:23.273Z2025-04-05T16:40:40.315ZS-EPMC101481533687176210.1016/j.jbc.2023.104582