Optimized orbitrap HCD for quantitative analysis of phosphopeptides View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2009-08

AUTHORS

Yi Zhang, Scott B. Ficarro, Shaojuan Li, Jarrod A. Marto

ABSTRACT

Despite the tremendous commercial success of radio frequency quadrupole ion traps for bottom-up proteomics studies, there is growing evidence that peptides decorated with labile post-translational modifications are less amenable to low-energy, resonate excitation MS/MS analysis. Moreover, multiplexed stable isotope reagents designed for MS/MS-based quantification of peptides rely on accurate and robust detection of low-mass fragments for all precursors. Collectively these observations suggest that beam-type or tandem in-space MS/MS measurements, such as that available on traditional triple quadrupole mass spectrometers, may provide beneficial figures of merit for quantitative proteomics analyses. The recent introduction of a multipole collision cell adjacent to an Orbitrap mass analyzer provides for higher energy collisionally activated dissociation (HCD) with efficient capture of fragment ions over a wide mass range. Here we describe optimization of various instrument and post-acquisition parameters that collectively provide for quantification of iTRAQ-labeled phosphorylated peptides isolated from complex cell lysates. Peptides spanning a concentration dynamic range of 100:1 are readily quantified. Our results indicate that appropriate parameterization of collision energy as a function of precursor m/z and z provides for optimal performance in terms of peptide identification and relative quantification by iTRAQ. Using this approach, we readily identify activated signaling pathways downstream of oncogenic mutants of Flt-3 kinase in a model system of human myeloid leukemia. More... »

PAGES

1425-1434

Identifiers

URI

http://scigraph.springernature.com/pub.10.1016/j.jasms.2009.03.019

DOI

http://dx.doi.org/10.1016/j.jasms.2009.03.019

DIMENSIONS

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PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/19403316


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