Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation
Research output: Contribution to journal › Journal article › Research › peer-review
Standard
Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation. / Nagaraj, Nagarjuna; D'Souza, Rochelle C J; Cox, Juergen; Olsen, Jesper Velgaard; Mann, Matthias.
In: Journal of Proteome Research, Vol. 9, No. 12, 03.12.2010, p. 6786-94.Research output: Contribution to journal › Journal article › Research › peer-review
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation
AU - Nagaraj, Nagarjuna
AU - D'Souza, Rochelle C J
AU - Cox, Juergen
AU - Olsen, Jesper Velgaard
AU - Mann, Matthias
PY - 2010/12/3
Y1 - 2010/12/3
N2 - Mass spectrometry (MS)-based proteomics now enables the analysis of thousands of phosphorylation sites in single projects. Among a wide range of analytical approaches, the combination of high resolution MS scans in an Orbitrap analyzer with low resolution MS/MS scans in a linear ion trap has proven to be particularly successful ("high-low" strategy). Here we investigate if the improved sensitivity of higher energy collisional dissociation (HCD) on an LTQ-Orbitrap Velos instrument allows a "high-high" strategy. A high resolution MS scan was followed by up to 10 HCD MS/MS scans, and we achieved cycle times of about 3 s making the method compatible with chromatographic time scales. Fragment mass accuracy increased about 50-fold compared to the "high-low" strategy. Unexpectedly, the HCD approach mapped up to 16,000 total phosphorylation sites in one day's measuring time--the same or better than the standard high-low strategy. Reducing the target values from a standard of 30,000 to 5000 ions did not severely affect identification rates but did decrease identification and localization scores for phosphorylation sites. We conclude that HCD in the new configuration is now a viable method for large-scale phosphoproteome analysis alongside collisional induced dissociation, (CID) and electron capture/transfer dissociation (ECD/ETD).
AB - Mass spectrometry (MS)-based proteomics now enables the analysis of thousands of phosphorylation sites in single projects. Among a wide range of analytical approaches, the combination of high resolution MS scans in an Orbitrap analyzer with low resolution MS/MS scans in a linear ion trap has proven to be particularly successful ("high-low" strategy). Here we investigate if the improved sensitivity of higher energy collisional dissociation (HCD) on an LTQ-Orbitrap Velos instrument allows a "high-high" strategy. A high resolution MS scan was followed by up to 10 HCD MS/MS scans, and we achieved cycle times of about 3 s making the method compatible with chromatographic time scales. Fragment mass accuracy increased about 50-fold compared to the "high-low" strategy. Unexpectedly, the HCD approach mapped up to 16,000 total phosphorylation sites in one day's measuring time--the same or better than the standard high-low strategy. Reducing the target values from a standard of 30,000 to 5000 ions did not severely affect identification rates but did decrease identification and localization scores for phosphorylation sites. We conclude that HCD in the new configuration is now a viable method for large-scale phosphoproteome analysis alongside collisional induced dissociation, (CID) and electron capture/transfer dissociation (ECD/ETD).
U2 - 10.1021/pr100637q
DO - 10.1021/pr100637q
M3 - Journal article
C2 - 20873877
VL - 9
SP - 6786
EP - 6794
JO - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 12
ER -
ID: 32355644