Gas-phase rearrangements do not affect site localization reliability in phosphoproteomics data sets

Abstract

Intramolecular transfer of phosphate during collision-induced dissociation (CID) in ion-trap mass spectrometers has recently been described. Because phosphorylation events are assigned to discrete serine, threonine, and tyrosine residues based on the presence of site-determining ions in MS/MS spectra, phosphate transfer may invalidate or confound site localization in published large-scale phosphorylation data sets. Here, we present evidence for the occurrence of this phenomenon using synthetic phosphopeptide libraries, specifically for doubly charged species. We found, however, that the extent of the transfer reaction was insufficient to cause localization of phosphorylation sites to incorrect residues. We further compared CID to electron-transfer dissociation (ETD) for site localization using synthetic libraries and a large-scale yeast phosphoproteome experiment. The agreement in site localization was >99.5 and 93%, respectively, suggesting that ETD-based site localization is no more reliable than CID. We conclude that intramolecular phosphate transfer does not affect the reliability of current or past phosphorylation data sets.

Figure 1

Schematic for intramolecular phosphate transfer reaction and phosphopeptide libraries used in this study. (A) Intramolecular phosphate transfer has been reported as a common occurrence in ion trap MS/MS spectra. When it occurs, the final spectrum may represent a composite of fragment ion peaks from the original and phosphate-transfer species, which could result in mislocalized phosphorylation events. (B) Based on the original sequences used by Palumbo and Reid , two libraries were created each with a known site and only one additional potential phosphate-transfer site. Each library was interrogated by LC- MS/MS techniques.

Figure 2

Evaluation of intramolecular phosphate transfer in ion trap MS/MS spectra. Each library was analyzed in triplicate by LC-MS/MS techniques in an LTQ Orbitrap XL mass spectrometer followed by database searching with Sequest and Ascore for site localization. CID MS/MS spectra (5427) were evaluated individually by keeping only the N most intense peaks (N=1 to 10) in every 100 m/z window. Only site-determining peaks matching to predicted b- or y-type ions from the correct (known) and incorrect (phosphate-transfer species) were counted. Doubly-charged (A), but not triply- or quadruply-charged (B,C) spectra contained evidence of more ions matched than expected by chance. *P<10⁻¹⁰ for both libraries compared to binomial using χ² test. Data are plotted as the mean from triplicate analysis of the two libraries with error bars at minimum and maximum values.

Figure 3

Intramolecular phosphate transfer does not appreciably affect site localization in synthetic peptide libraries. Each library was analyzed in triplicate by LC-MS/MS techniques where back-to-back CID and ETD spectra were collected for each selected phosphopeptide ion. Peptide identifications were filtered to <0.5% FDR. Data are shown as the mean of both libraries (triplicate analysis summed) with error bars at the minimum and maximum values. (A) ETD identified 10.1 % more peptides from these highly basic libraries. (B) CID and ETD are suited to different populations of charge states. (C) The fraction of incorrect site assignments either due to chance or to intramolecular phosphate transfer is very small. ETD is not significantly more reliable than CID at site localization in these known-site synthetic peptide libraries.

Figure 4

Comparison of CID and ETD fragmentation in a large-scale yeast phosphorylation analysis. (A) Workflow for phosphoproteomic evaluation. Yeast phosphopeptides were enriched via the SCX-IMAC approach. Back-to-back ETD and CID spectra were acquired for each selected phosphopeptide ion in 15 SCX fractions. (B) CID is considerably more efficient than ETD for large scale phosphoproteomics at a 1% FDR. (C) Among overlapping peptides (1387), the vast majority of localized sites (P<0.05) agreed between CID and ETD (93.2 %). (D) Phosphopeptides identified in each SCX fraction. ETD can be leveraged to identify more phosphopeptides in late eluting SCX fractions.