Protocol abstract
Nature Protocols 4, - 870 - 877 (2009)
Published online: 14 May 2009 | doi:10.1038/nprot.2009.57
Subject Categories: Biochemistry and protein analysis | Computational and theoretical biology | Genomics and proteomics | Spectroscopy and structural analysis
A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS)
Richard D Unwin1, John R Griffiths1 & Anthony D Whetton1
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.
- Stem Cell and Leukaemia Proteomics Laboratory, Faculty of Medicine and Human Sciences, University of Manchester, Manchester, UK.
Correspondence to: Richard D Unwin1 e-mail: r.unwin@manchester.ac.uk
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