Optimized orbitrap HCD for quantitative analysis of phosphopeptides

  • Yi Zhang
  • Scott B. Ficarro
  • Shaojuan Li
  • Jarrod A. Marto
Focus: The Orbitrap

DOI: 10.1016/j.jasms.2009.03.019

Cite this article as:
Zhang, Y., Ficarro, S.B., Li, S. et al. J Am Soc Mass Spectrom (2009) 20: 1425. doi:10.1016/j.jasms.2009.03.019
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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.

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Supplementary material

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13361_2011_200801425_MOESM2_ESM.pdf (425 kb)
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Copyright information

© American Society for Mass Spectrometry 2009

Authors and Affiliations

  • Yi Zhang
    • 1
  • Scott B. Ficarro
    • 1
  • Shaojuan Li
    • 1
  • Jarrod A. Marto
    • 1
  1. 1.Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonUSA

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