Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment. / Kelstrup, Christian D; Hekmat, Omid; Francavilla, Chiara; Olsen, Jesper V.

In: Journal of Proteome Research, Vol. 10, No. 7, 01.07.2011, p. 2937-48.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Kelstrup, CD, Hekmat, O, Francavilla, C & Olsen, JV 2011, 'Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment', Journal of Proteome Research, vol. 10, no. 7, pp. 2937-48. https://doi.org/10.1021/pr200154t

APA

Kelstrup, C. D., Hekmat, O., Francavilla, C., & Olsen, J. V. (2011). Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment. Journal of Proteome Research, 10(7), 2937-48. https://doi.org/10.1021/pr200154t

Vancouver

Kelstrup CD, Hekmat O, Francavilla C, Olsen JV. Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment. Journal of Proteome Research. 2011 Jul 1;10(7):2937-48. https://doi.org/10.1021/pr200154t

Author

Kelstrup, Christian D ; Hekmat, Omid ; Francavilla, Chiara ; Olsen, Jesper V. / Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment. In: Journal of Proteome Research. 2011 ; Vol. 10, No. 7. pp. 2937-48.

Bibtex

@article{0198dc999bd74b25894abbd8e59d62a4,
title = "Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment",
abstract = "Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site assignments in large-scale phosphoproteomics data sets. To improve methods for site localization, we made use of a synthetic phosphopeptide library and SILAC-labeled peptides from whole cell lysates and analyzed these with high-resolution tandem mass spectrometry on an LTQ Orbitrap Velos. We validated gas-phase phosphate rearrangement reactions during collision-induced dissociation (CID) and used these spectra to devise a quantitative filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissociation (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements observed above noise level. Analyzing a large set of HCD spectra of SILAC-labeled phosphopeptides, we identified a novel fragmentation mechanism that generates a phosphorylation site-specific neutral loss derived x-ion, which directly pinpoints the phosphorylated residue. Together, these findings significantly improve phosphorylation site localization confidence.",
author = "Kelstrup, {Christian D} and Omid Hekmat and Chiara Francavilla and Olsen, {Jesper V}",
year = "2011",
month = jul,
day = "1",
doi = "10.1021/pr200154t",
language = "English",
volume = "10",
pages = "2937--48",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "7",

}

RIS

TY - JOUR

T1 - Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment

AU - Kelstrup, Christian D

AU - Hekmat, Omid

AU - Francavilla, Chiara

AU - Olsen, Jesper V

PY - 2011/7/1

Y1 - 2011/7/1

N2 - Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site assignments in large-scale phosphoproteomics data sets. To improve methods for site localization, we made use of a synthetic phosphopeptide library and SILAC-labeled peptides from whole cell lysates and analyzed these with high-resolution tandem mass spectrometry on an LTQ Orbitrap Velos. We validated gas-phase phosphate rearrangement reactions during collision-induced dissociation (CID) and used these spectra to devise a quantitative filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissociation (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements observed above noise level. Analyzing a large set of HCD spectra of SILAC-labeled phosphopeptides, we identified a novel fragmentation mechanism that generates a phosphorylation site-specific neutral loss derived x-ion, which directly pinpoints the phosphorylated residue. Together, these findings significantly improve phosphorylation site localization confidence.

AB - Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site assignments in large-scale phosphoproteomics data sets. To improve methods for site localization, we made use of a synthetic phosphopeptide library and SILAC-labeled peptides from whole cell lysates and analyzed these with high-resolution tandem mass spectrometry on an LTQ Orbitrap Velos. We validated gas-phase phosphate rearrangement reactions during collision-induced dissociation (CID) and used these spectra to devise a quantitative filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissociation (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements observed above noise level. Analyzing a large set of HCD spectra of SILAC-labeled phosphopeptides, we identified a novel fragmentation mechanism that generates a phosphorylation site-specific neutral loss derived x-ion, which directly pinpoints the phosphorylated residue. Together, these findings significantly improve phosphorylation site localization confidence.

U2 - 10.1021/pr200154t

DO - 10.1021/pr200154t

M3 - Journal article

C2 - 21526838

VL - 10

SP - 2937

EP - 2948

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 7

ER -

ID: 33743697