期刊
PROTEOMICS
卷 6, 期 -, 页码 S16-S27出版社
WILEY
DOI: 10.1002/pmic.200500407
关键词
Linear ion trap; Nuclear factor of activated T cells; Phosphorylation
资金
- Fundacion Ramon Areces
- RECAVA from Ministerio de Sanidad y Consumo
- Junta de Comunidades de Castilla- La Mancha (JCCM)
- Centro Nacional de Investigaciones Cardiovasculares (CNIC)
- [CICYT BIO2003-01805]
- [CAM 08.5/0065.1/2001]
- [CAM GR/SAL/0141/2004]
- [SAF 2003-02920]
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.
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