Combined Pulsed-Q dissociation and electron transfer dissociation for identification and quantitation of iTRAQ–labeled phosphopeptides
Multiplex isobaric tags for relative and absolute quantification (iTRAQ) enable high-throughput quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. A challenging but highly promising application is to analyze iTRAQ-labeled peptides using a sensitive linear ion trap mass spectrometer (LTQ-MS) and pulsed Q dissociation (PQD), a form of ion trap collision activated dissociation (CAD) designed to allow detection of low mass-to-charge fragment ions. Electron dissociation transfer (ETD), on the other hand, is complementary to PQD and is especially useful for sequencing peptides containing post-translational modifications (PTMs). Here, we developed an integrated workflow for robust and accurate quantitative identification of iTRAQ labeled phosphopeptides that integrates the PQD and ETD fragmentation methods together with PQD optimization, data management and bioinformatics tools. Analysis of the phosphoproteome of human fibroblast cells demonstrated that this hybrid mode is superior to either PQD or ETD alone for phosphopeptide identification and quantitation. The combined PQD/ETD approach can qualitatively identify additional phosphopeptides than ETD alone and PQD information can provide better quantitation of ETD identified iTRAQ-labeled phosphopeptides.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 958485
- Report Number(s):
- PNNL-SA-63701; 24698; 400412000; KP1602020; TRN: US201002%%56
- Journal Information:
- Analytical Chemistry, 81(10):4137-4143, Vol. 81, Issue 10
- Country of Publication:
- United States
- Language:
- English
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