A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS).
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.
Unwin
Richard D
RD
Stem Cell and Leukaemia Proteomics Laboratory, Faculty of Medicine and Human Sciences, University of Manchester, Manchester, UK. r.unwin@manchester.ac.uk
Griffiths
John R
JR
Whetton
Anthony D
AD
D000595:Amino Acid Sequence; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008991:Monitoring, Physiologic / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D020543:Proteome; D040901:Proteomics / Q000379:methods; D012106:Research / Q000639:trends; D012680:Sensitivity and Specificity; D013816:Thermodynamics
D010446:Peptide Fragments; D010455:Peptides; D020543:Proteome