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/MSbased 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. [4][5][6], extended mass range capability [7], use of helium buffer gas [3], and automated control of trapped ion populations [8], all combined to catapult trap-based instruments to the forefront of proteomics research. Concomitant advances in CPU and embedded systems provided for rapid and automated control of instrument parameters and yielded heretofore unattainable throughput for peptide sequence analysis via LC-MS/MS. Today, several years past the emergence of proteomics as a field of active research [9], it is clear that "typical" tryptic peptides are very amenable to sequence analysis via low-energy MS/MS. More recently, the introduction of hybrid geometries [10 -12] and other instrument configurations [13,14], along with protocols for enrichment of post-translationally modified peptides [15,16], have dramatically increased the experimental dynamic range of proteomics analyses. Collectively, these results have revealed that peptides decorated with labile modifications often are not well behaved under low-energy MS/MS conditions. For example, facile loss of phosphoric acid from the side chains of serine-and threoninephosphorylated peptides often limits the available sequence-specific information contained in associated MS/MS spectra [17,18]. Interestingly, recent reports [19,20] suggest that higher energy, or "triple quadrupole like," MS/MS can provide improved sequence analysis, particularly for phosphorylated peptides. In the work repo...

Online Nanoflow Multidimensional Fractionation for High Efficiency Phosphopeptide Analysis

“…Moreover, the results in Fig. 4 represent a nearly 10-fold increase in the number of phosphorylation sites detected as compared with a recent study from our lab in which we used an offline RP fractionation scheme (five fractions total) to analyze FLT3 signaling in the same cell system, under the same culture conditions (68). The efficiency of our RP-SAX-RP platform is further evidenced by the fact that our data compare favorably in scale (4586 phosphorylation sites) to that in a recent report from Choudhary et al (14700 unique phosphorylation sites) (69), while consuming some 25-fold less cell lysate (400 g versus 10 mg).…”

A Robust Error Model for iTRAQ Quantification Reveals Divergent Signaling between Oncogenic FLT3 Mutants in Acute Myeloid Leukemia

“…The low mass cutoff for HCD scans was set at 100 amu. The data files were processed with in-house software to separate CAD and HCD scans into two Mascot-searchable files as described previously (24). The two files were submitted to Mascot (v2.1) and searched against all 55,824 entries in a database that consisted of human FLT3 and the NCBI RefSeq mouse protein database (released on April 3, 2006).…”

Quantitative Proteomics Using iTRAQ Labeling and Mass Spectrometry

“…This meant that traditionally, iTRAQ TM -based quantification was not possible using an ion trap or hybrid instrument containing an ion trap such as the LTQ-Orbitrap. Recently developed fragmentation methods now make it possible to perform iTRAQ TM -based quantification on an LTQ-Orbitrap and include Pulsed Q Dissociation (PQD) (Bantscheff et al, 2008) and higher energy C-trap dissociation (HCD) (Zhang et al, 2009). Both fragmentation methods are less suited for protein identification at a proteomic scale than CID fragmentation, but when combined with CID, HCD allows sensitive and accurate iTRAQ TM quantification of whole proteomes (Köcher et al, 2009).…”