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Updated: Aug 10, 2026
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Systematic characterization of phosphorylation sites in NFATc2 by linear ion trap mass spectrometry
Margarita Villar1, Inmaculada Ortega-Pérez, Felipe Were
1Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.
Abstract:
Members of the nuclear factor of activated T cells (NFAT) family of transcription factors regulate transcription of genes involved in the function of many different cellular systems. Activity of NFAT proteins is regulated by a complex interplay of phosphorylation events that are still poorly understood. In this work, we take advantage of the high scanning speed of the linear ion trap to develop a method to make a systematic characterization of NFATc2 phosphorylation. The method is based on the simultaneous monitoring of all tryptic peptides that can be detected by MS and contain potential phosphorylation sites. By this approach, we detected six NFATc2 phosphorylation sites by c-Jun NH2-terminal kinase (JNK) in vitro in only one experiment; a further site was also identified by performing digestion in solution. Using this approach, we have also characterized five basal phosphorylation sites in NFATc2 protein expressed in HEK cell cultures. Two of these NFATc2 phosphorylation sites in vivo have not been described before. The simplicity and sensitivity of this technique, which can be applied to any potential modification, makes it particularly attractive for the systematic detection of post-translational modifications of specific target proteins both in vitro and in vivo.
Insights
This study presents a new mass spectrometry method for systematically identifying nuclear factor of activated T cells 2 (NFATc2) phosphorylation sites. The technique efficiently detected multiple in vitro and in vivo phosphorylation sites, including novel ones.
Area of Science:
- Proteomics
- Molecular Biology
- Cellular Signaling
Background:
- Nuclear factor of activated T cells (NFAT) proteins are crucial transcription factors regulating diverse cellular functions.
- NFAT protein activity is modulated by phosphorylation, a process that remains incompletely understood.
- Systematic characterization of NFATc2 phosphorylation is essential for deciphering its regulatory mechanisms.
Purpose of the Study:
- To develop and apply a high-throughput mass spectrometry (MS) method for systematic characterization of NFATc2 phosphorylation.
- To identify novel in vitro and in vivo phosphorylation sites on NFATc2.
- To establish a sensitive technique for detecting post-translational modifications.
Main Methods:
- Utilized a linear ion trap mass spectrometer for rapid scanning and simultaneous monitoring of tryptic peptides.
- Employed a method for detecting all detectable tryptic peptides containing potential phosphorylation sites.
- Performed in vitro phosphorylation analysis using c-Jun NH2-terminal kinase (JNK) and in vivo analysis of NFATc2 expressed in HEK cell cultures.
Main Results:
- Successfully identified six NFATc2 phosphorylation sites induced by JNK in vitro in a single experiment.
- Identified an additional NFATc2 phosphorylation site through solution digestion.
- Characterized five basal phosphorylation sites on NFATc2 in vivo, with two sites being previously undescribed.
- Demonstrated the method's applicability to both in vitro and in vivo samples.
Conclusions:
- The developed MS-based method enables efficient and systematic characterization of NFATc2 phosphorylation.
- This approach identified novel phosphorylation sites, advancing the understanding of NFATc2 regulation.
- The technique's simplicity and sensitivity make it valuable for systematic detection of post-translational modifications in various biological contexts.
