Abstract
The application of a targeted mass spectrometric workflow to the sensitive identification of post-translational modifications is described. This protocol employs multiple reaction monitoring (MRM) to search for all putative peptides specifically modified in a target protein. Positive MRMs trigger an MS/MS experiment to confirm the nature and site of the modification. This approach, termed MIDAS (MRM-initiated detection and sequencing), is more sensitive than approaches using neutral loss scanning or precursor ion scanning methodologies, due to a more efficient use of duty cycle along with a decreased background signal associated with MRM. We describe the use of MIDAS for the identification of phosphorylation, with a typical experiment taking just a couple of hours from obtaining a peptide sample. With minor modifications, the MIDAS method can be applied to other protein modifications or unmodified peptides can be used as a MIDAS target.
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This study was supported by Leukaemia Research UK and BBSRC.
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Unwin, R., Griffiths, J. & Whetton, A. A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS). Nat Protoc 4, 870–877 (2009). https://doi.org/10.1038/nprot.2009.57
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DOI: https://doi.org/10.1038/nprot.2009.57
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