Comparative assessment of site assignments in CID and electron transfer dissociation spectra of phosphopeptides discloses limited relocation of phosphate groups.
Mol Cell Proteomics
; 9(10): 2140-8, 2010 Oct.
Article
em En
| MEDLINE
| ID: mdl-20233845
ABSTRACT
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 â¼75% 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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Fosfatos
/
Fosfopeptídeos
Idioma:
En
Revista:
Mol Cell Proteomics
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Holanda