Towards liquid chromatography time-scale peptide sequencing and characterization of post-translational modifications in the negative-ion mode using electron detachment dissociation tandem mass spectrometry.
Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards post-translational modifications (PTMs) and produces predictable and interpretable fragment ion types (a., x ions). However, EDD is considered an inefficient fragmentation technique and has not yet been implemented in large-scale peptide characterization strategies. We successfully increased the EDD fragmentation efficiency (up to 9%), and demonstrate for the first time the utility of EDD-MS/MS in liquid chromatography time-scale experiments. Peptides and phosphopeptides were analyzed in both positive- and negative-ion mode using electron capture/transfer dissociation (ECD/ETD) and EDD in comparison. Using approximately 1 pmol of a BSA tryptic digest, LC-EDD-MS/MS sequenced 14 peptides (27% aa sequence coverage) and LC-ECD-MS/MS sequenced 19 peptides (39% aa sequence coverage). Seven peptides (18% aa sequence coverage) were sequenced by both EDD and ECD. The relative small overlap of identified BSA peptides demonstrates the complementarity of the two dissociation modes. Phosphopeptide mixtures from three trypsin-digested phosphoproteins were subjected to LC-EDD-MS/MS resulting in the identification of five phospho-peptides. Of those, one was not found in a previous study using a similar sample and LC-ETD-MS/MS in the positive-ion mode. In this study, the ECD fragmentation efficiency (15.7% av.) was superior to the EDD fragmentation efficiency (3.6% av.). However, given the increase in amino acid sequence coverage and extended PTM characterization the new regime of EDD in combination with other ion-electron fragmentation techniques in the positive-ion mode is a step towards a more comprehensive strategy of analysis in proteome research.
Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense, Denmark. Frankk@bmb.sdu.dk
Frank
F
Kjeldsen
Ole B
OB
Hørning
Søren S
SS
Jensen
Anders M B
AM
Giessing
Ole N
ON
Jensen
D000595:Amino Acid Sequence; D004583:Electrons; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared; D053719:Tandem Mass Spectrometry
D010455:Peptides; D010748:Phosphopeptides