Phosphotyrosine

The full story is described in this publication: Voinov VG, Bennett SE, Beckman JS, Barofsky DF. ECD of tyrosine phosphorylation in a triple quadrupole mass spectrometer with a radio-frequency-free electromagnetostatic cell. J Am Soc Mass Spectrom. 2014 Oct;25(10):1730-8. PMC4163116.  

Analysis of post-translational modifications such as phosphorylation using CID is challenging because the phosphate is easily and unpredictably dislodged by collisions.  Thus, phosphorylation is difficult to quantify by CID.  

Here is an example of a phosphopeptide fragmented by CID.  

1598 2+ CID Agilent

Traditional Electron Capture Dissociation on a Fourier-Transform Ion-Cyclotron Resonance Mass spectrometer

ECD is well known to preserve phosphorylation modifications, but the technique has difficulties working with doubly charged peptides.  The phosphotyrosine is known to hold fragments of the peptide together via strong ionic interactions.  Hence the fragmented peptides do not separate in the mass spectrometery.  This spectrum was collected on an FT-ICR and required over 10 minutes to acquire.  

1598 2+ ECD Bruker FT

Our ECD cell installed in a Triple Quad produced the following spectrum with only a few scans.  

1598 2+ ECD Agilent

 The hot filament in our cell most likely produces infrared radiation that dissociated the sticky fragments held together by the phosphogroup.  

This infrared radiation however does not disturb the phosphotyrosine bond, leaving a clean spectrum that could allow automated de novo sequencing.  

White-hot Loop Filament used in our ECD cell.  

White hot loop emitter.001