Related Experiment Video
Updated: Sep 28, 2026
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning
Hanno Steen1, Akhilesh Pandey, Jens S Andersen
1Harvard Medical School, Department of Cell Biology, 240 Longwood Avenue, Boston, MA 02115, USA. hanno_steen@hms.harvard.edu
Abstract:
Signal transduction pathways involve cascades of events, such as formation of second messengers and protein complexes that alter the activities of proteins. This can ultimately lead to changes in gene expression in response to the stimuli. Reversible phosphorylation of proteins is an important mechanism for activating or inhibiting enzymes and for the assembly of multiprotein complexes. Here, we describe a mass spectrometry-based phosphotyrosine-specific immonium ion scanning (PSI scanning) method for selective detection of tyrosine-phosphorylated peptides. Once the tyrosine-phosphorylated peptides are identified, they can be directly sequenced in the same experiment to localize the phosphorylation site. We provide protocols for isolation and preparation of samples for analysis, and detailed instructions for operation of a quadrupole time-of-flight (TOF) mass spectrometer for this method. Because of its simplicity and specificity, PSI scanning is likely to become an important tool in proteomic studies of pathways involving tyrosine phosphorylation.
Insights
We developed a mass spectrometry method called PSI scanning to detect tyrosine-phosphorylated peptides. This technique allows for precise localization of phosphorylation sites, aiding proteomic studies of signal transduction pathways.
Area of Science:
- Proteomics
- Molecular Biology
- Biochemistry
Background:
- Signal transduction pathways regulate cellular responses through complex cascades.
- Protein phosphorylation, particularly tyrosine phosphorylation, is a key regulatory mechanism in these pathways.
- Identifying specific phosphorylation sites is crucial for understanding enzyme activity and protein complex assembly.
Purpose of the Study:
- To introduce a novel mass spectrometry-based method for selective detection of tyrosine-phosphorylated peptides.
- To enable direct sequencing of these peptides for phosphorylation site localization within the same experiment.
- To provide comprehensive protocols for sample preparation and instrument operation for this technique.
Main Methods:
- Development of a phosphotyrosine-specific immonium ion scanning (PSI scanning) technique.
- Utilizing quadrupole time-of-flight (TOF) mass spectrometry for peptide analysis.
- Implementing protocols for sample isolation, preparation, and mass spectrometry operation.
Main Results:
- Demonstrated the ability of PSI scanning to selectively detect tyrosine-phosphorylated peptides.
- Showcased the capacity for direct sequencing to pinpoint phosphorylation sites.
- Established protocols for effective implementation of the PSI scanning method.
Conclusions:
- PSI scanning offers a simple and specific approach for identifying tyrosine-phosphorylated peptides.
- This method is poised to become a valuable tool in proteomic research, particularly for studying tyrosine phosphorylation in signal transduction.
- The developed protocols facilitate the application of PSI scanning in diverse proteomic studies.
More Related Videos
Related Concept Videos
02:54Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
01:26Receptor Tyrosine Kinases