Creese and Cooper
Page 3
Sponsored Document Sponsored Document Sponsored Document
The introduction of data dependent on-line liquid chromatography (LC)-ECD MS/MS has
allowed automated analysis of complex protein samples. We have recently demonstrated data
dependent nano-LC ECD MS/MS for the identification of the protein ROR2 isolated from
human chondrocytes [33]. Because ECD and CID produce different backbone fragments, they
are complimentary methods for peptide identification and combined provide a large amount
of information. Zubarev and coworkers have developed a method performing both ECD and
CID on all eluting peptides [34-38]. This combined approach provides information about the
relationship between b/y and c/z ∙ fragments, i.e.; the “golden rules” [39]. Recent studies in our
laboratory have shown that on-line LC-ECD MS/MS identifies peptides with greater
confidence than on-line LC-CID MS/MS in terms of the extent of sequence tag [17].
Nevertheless, the time scale for ECD remains considerably longer than that for CID. The result
is that fewer peptides are selected for fragmentation in an LC-ECD MS/MS analysis. This is
particularly pertinent in the case of phosphopeptide analysis; phosphopeptides tend to exist at
lower stoichiometry than their unmodified counterparts and can be overlooked. Enrichment of
protein digests for phosphopeptides by use of titanium dioxide [40] or zirconium dioxide
[41] affinity chromatography alleviates the problem to some extent. We have developed two
targeted LC-ECD approaches that focus ECD time on phosphopeptides. In neutral loss (NL)
ECD [42], protein digests are separated by on-line LC. All eluting peptides are subjected to
CID. If a neutral loss corresponding to loss of phosphoric acid (-98 Da) is observed in the CID
mass spectrum, ECD of the precursor ion is triggered. In targeted ECD [32], the protein digest
is first analyzed by LC-CID MS/MS and putative phosphopeptides identified by mass. An LC-
ECD MS/MS analysis is then performed in which only potential phosphopeptides are
investigated. These methods combine the high scan rate of CID with the improved peptide
sequence coverage and the retention of PTMs of ECD.
Here, we investigate the effect of site and frequency of peptide phosphorylation on electron
capture dissociation behavior. We have compared the ECD of eight synthetic phosphopeptides
(APLSFRGSLPKSYVK) (one unmodified, three singly-phosphorylated, three-doubly
phosphorylated, and one triply-phosphorylated) over a range of ECD cathode potentials. We
have also investigated the behavior of two tryptic phosphopeptides, one from α-S1-casein
(YKVPQLEIVPNpSAEER) and one from β-casein (FQpSEEQQQTEDELQDK), and their
nonphosphorylated counterparts. The results show that for doubly-charged peptide precursor
ions the presence of phosphorylation has a deleterious effect on ECD sequence coverage. The
fragmentation pattern observed suggests this is the result of the deprotonated phospho-group
forming salt bridges with protonated side chains of basic amino acid residues. Increasing the
ECD electron energy (by decreasing the ECD cathode potential) significantly improves
sequence coverage. It is postulated that the deposition of additional energy facilitates cleavage
of the noncovalent bonds enabling detection of the backbone fragments, in a similar manner
to activated ion ECD [43]. Improved sequence coverage can also be obtained by ECD of triply-
charged precursor ions. The tryptic peptides showed improved sequence coverage with
increasing electron energy for both phosphopeptides and their unmodified counterparts. These
results suggest that for these peptides the phospho-group was not directly involved in
noncovalent bonding but that its presence altered the peptide conformation and intramolecular
bonding.
Experimental
Preparation of Synthetic Peptides
A suite of eight synthetic peptides (APLSFRGSLPKSYVK: one unmodified, three singly-
phosphorylated, three doubly-phosphorylated, and one triply-phosphorylated) were
synthesized by Alta Biosciences (Birmingham, UK) and used without further purification. The
peptides were diluted to 2 pmol∕μL in methanol (Fisher Scientific, Leicestershire, UK):water
(J. T. Baker, Deventer, The Netherlands) (75:25) + 1% formic acid (Fisher Scientific). α-Casein
Published as: J Am Soc Mass Spectrom. 2008 September ; 19(9): 1263-1274.