Comparative assessment of site assignments in CID and electron transfer dissociation spectra of phosphopeptides discloses limited relocation of phosphate groups.
In large scale mass spectrometry-based phosphoproteomics, a current bottleneck is the unambiguous assignment of the phosphorylation site within the peptide. An additional problem is that it has been reported that under conditions wherein peptide ions are collisionally activated the phosphate group may migrate to a nearby phosphate group acceptor, thus causing ambiguity in site assignment. Here, we generated and analyzed a statistically significant number of phosphopeptides. Starting with a human cell lysate, we obtained via strong cation exchange fractionation nearly pure phosphopeptide pools from trypsin and Lys-N digestions. These pools were subjected to nano-LC-MS using an Orbitrap mass spectrometer that is equipped with both CID and electron transfer dissociation with supplemental activation (ETcaD) functionality. We configured a method to obtain sequentially both ETcaD and CID spectra for each peptide ion. We exploited the resistant nature of ETcaD toward rearrangement of phosphate groups to evaluate whether there is potentially phosphate group relocation occurring during CID. We evaluated a number of peptide and spectral annotation properties and found that for ∼75% of the sequenced phosphopeptides the assigned phosphosite was unmistakably identical for both the ETcaD and CID spectra. For the remaining 25% of the sequenced phosphopeptides, we also did not observe evident signs of relocation, but these peptides exhibited signs of ambiguity in site localization, predominantly induced by factors such as poor fragmentation, sequences causing inefficient fragmentation, and generally poor spectrum quality. Our data let us derive the conclusion that both for trypsin- and Lys-N-generated peptides there is little relocation of phosphate groups occurring during CID.
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Nikolai
N
Mischerikow
A F Maarten
AF
Altelaar
J Daniel
JD
Navarro
Shabaz
S
Mohammed
Albert J R
AJ
Heck
D004579:Electron Transport; D013058:Mass Spectrometry; D010710:Phosphates / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation
D010710:Phosphates; D010748:Phosphopeptides