title: Optimized Orbitrap HCD for quantitative analysis of phosphopeptides.
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.
authors.affiliation: Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115-6084, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Scott B
authors.initials: SB
authors.lastname: Ficarro
authors.forename: Shaojuan
authors.initials: S
authors.lastname: Li
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
mesh_terms: D000465:Algorithms; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D010748:Phosphopeptides