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Sci. STKE, 15 October 2002
Vol. 2002, Issue 154, p. pl16
[DOI: 10.1126/stke.2002.154.pl16]

PROTOCOLS

Analysis of Tyrosine Phosphorylation Sites in Signaling Molecules by a Phosphotyrosine-Specific Immonium Ion Scanning Method

Hanno Steen1, Akhilesh Pandey2, Jens S. Andersen3, and Matthias Mann3*

1Harvard Medical School, Department of Cell Biology, 240 Longwood Avenue, Boston, MA 02115, USA. E-mail:hanno_steen{at}hms.harvard.edu.
2McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University, 600 North Wolfe Street, Baltimore, MD 21287, USA. E-mail:pandey{at}jhmi.edu.
3The Protein Interaction Laboratory, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. E-mail: jens.andersen{at}bmb.sdu.dk.

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.

*Corresponding author. E-mail, mann@bmb.sdu.dk

Citation: H. Steen, A. Pandey, J. S. Andersen, M. Mann, Analysis of Tyrosine Phosphorylation Sites in Signaling Molecules by a Phosphotyrosine-Specific Immonium Ion Scanning Method. Sci. STKE 2002, pl16 (2002).

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