Pulsed Q Dissociation Research Articles

Combined pulsed-Q dissociation and electron transfer dissociation for identification and quantification of iTRAQ-labeled phosphopeptides.

Here, we report a new approach that integrates pulsed Q dissociation (PQD) and electron transfer dissociation (ETD) techniques for confident and quantitative identification of iTRAQ-labeled phosphopeptides. The use of isobaric tags for relative and absolute quantification enables a high-throughput quantification of peptides via reporter ion signals in the low m/z range of tandem mass spectra. PQD, a form of ion trap collision activated dissociation, allows for detection of low mass-to-charge fragment ions, and electron transfer dissociation is especially useful for sequencing peptides that contain post-translational modifications. Analysis of the phosphoproteome of human fibroblast cells using a sensitive linear ion trap mass spectrometer demonstrated that this hybrid approach improves both identification and quantification of phosphopeptides. ETD improved phosphopeptide identification, while PQD provides improved quantification of iTRAQ-labeled phosphopeptides.

Differential Protein Expression Analysis Using Stable Isotope Labeling and PQD Linear Ion Trap MS Technology

An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17beta-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of <4%. The reproducibility of protein identifications was approximately 60%-67% between duplicate, and approximately 50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P < 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate approximately 2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, approximately 16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up- or down-regulated.

Comparison of Infrared Multiphoton Dissociation and Collision-Induced Dissociation of Supercharged Peptides in Ion Traps

The number and types of diagnostic ions obtained by infrared multiphoton dissociation (IRMPD) and collision-induced dissociation (CID) were evaluated for supercharged peptide ions created by electrospray ionization of solutions spiked with m-nitrobenzyl alcohol. IRMPD of supercharged peptide ions increased the sequence coverage compared with that obtained by CID for all charge states investigated. The number of diagnostic ions increased with the charge state for IRMPD; however, this trend was not consistent for CID because the supercharged ions did not always yield the greatest number of diagnostic ions. Significantly different fragmentation pathways were observed for the different charge states upon CID or IRMPD with the latter yielding far more immonium ions and often fewer uninformative ammonia, water, and phosphoric acid neutral losses. Pulsed-Q dissociation resulted in an increase in the number of internal product ions, a decrease in sequence-informative ions, and reduced overall ion abundances. The enhanced sequence coverage afforded by IRMPD of supercharged ions was demonstrated for a variety of model peptides, as well as for a tryptic digest of cytochrome c.

Hybridization of Pulsed-Q Dissociation and Collision-Activated Dissociation in Linear Ion Trap Mass Spectrometer for iTRAQ Quantitation

Coupling of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) to a sensitive linear ion trap (LTQ) mass spectrometer (MS) is a challenging, but highly promising approach for quantitative high-throughput proteomic profiling. Integration of the advantages of pulsed-Q dissociation (PQD) and collision-activated dissociation (CAD) fragmentation methods into a PQD-CAD hybrid mode, together with PQD optimization and data manipulation with a bioinformatics algorithm, resulted in a robust, sensitive and accurate iTRAQ quantitative proteomic workflow. The workflow was superior to the default PQD setting when profiling the proteome of a gastric cancer cell line, SNU5. Taken together, we established an optimized PQD-CAD hybrid workflow in LTQ-MS for iTRAQ quantitative proteomic profiling that may have wide applications in biological and biomedical research.

Robust and Sensitive iTRAQ Quantification on an LTQ Orbitrap Mass Spectrometer

Isobaric stable isotope tagging reagents such as tandem mass tags or isobaric tags for relative and absolute quantification enable multiplexed quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. Until recently, the poor recovery of low mass fragments observed in tandem mass spectra acquired on ion trap mass spectrometers precluded the use of these reagents on this widely available instrument platform. The Pulsed Q Dissociation (PQD) technique allows negotiating this limitation but suffers from poor fragmentation efficiency, which has raised doubts in the community as to its practical utility. Here we show that by carefully optimizing instrument parameters such as collision energy, activation Q, delay time, ion isolation width, number of microscans, and number of trapped ions, low m/z fragment ion intensities can be generated that enable accurate peptide quantification at the 100 amol level. Side by side comparison of PQD on an LTQ Orbitrap with CID on a five-year old Q-Tof Ultima using complex protein digests shows that whereas precision of quantification of 10-15% can be achieved by both approaches, PQD quantifies twice as many proteins. PQD on an LTQ Orbitrap also outperforms "higher energy collision induced dissociation" on the same instrument using the recently introduced octapole collision cell in terms of lower limit of quantification. Finally, we demonstrate the significant analytical potential of iTRAQ quantification using PQD on an LTQ Orbitrap by quantitatively measuring the kinase interaction profile of the small molecule drug imatinib in K-562 cells. This article gives practical guidance for the implementation of PQD, discusses its merits, and for the first time, compares its performance to higher energy collision-induced dissociation.

Peptide and Protein Quantification Using iTRAQ with Electron Transfer Dissociation

Electron transfer dissociation (ETD) has become increasingly used in proteomic analyses due to its complementarity to collision-activated dissociation (CAD) and its ability to sequence peptides with post-translation modifications (PTMs). It was previously unknown, however, whether ETD would be compatible with a commonly employed quantification technique, isobaric tags for relative and absolute quantification (iTRAQ), since the fragmentation mechanisms and pathways of ETD differ significantly from CAD. We demonstrate here that ETD of iTRAQ labeled peptides produces c- and z-type fragment ions as well as reporter ions that are unique from those produced by CAD. Exact molecular formulas of product ions were determined by ETD fragmentation of iTRAQ-labeled synthetic peptides followed by high mass accuracy orbitrap mass analysis. These experiments revealed that ETD cleavage of the N-C(alpha) bond of the iTRAQ tag results in fragment ions that could be used for quantification. Synthetic peptide work demonstrates that these fragment ions provide up to three channels of quantification and that the quality is similar to that provided by beam-type CAD. Protein standards were used to evaluate peptide and protein quantification of iTRAQ labeling in conjunction with ETD, beam-type CAD, and pulsed Q dissociation (PQD) on a hybrid ion trap-orbitrap mass spectrometer. For reporter ion intensities above a certain threshold all three strategies provided reliable peptide quantification (average error <10%). Approximately 36%, 8%, and 16% of scans identified fall below this threshold for ETD, HCD, and PQD, respectively. At the protein level, average errors were 2.3%, 1.7%, and 3.6% for ETD, HCD, and PQD, respectively.

Identification of carbonylated proteins from enriched rat skeletal muscle mitochondria using affinity chromatography‐stable isotope labeling and tandem mass spectrometry

We describe a strategy for the identification of carbonylated proteins from complex protein mixtures that combines biotin hydrazide labeling of protein carbonyl groups, avidin affinity chromatography, multiplexed iTRAQ reagent stable isotope labeling, and analysis using pulsed Q dissociation (PQD) operation on an LTQ linear ion trap mass spectrometer. This strategy provided the ability to distinguish biotin hydrazide labeled, avidin purified, carbonylated proteins from non-carbonylated background proteins with affinity for the avidin column, derived from a control sample. Applying this strategy to the identification of crudely enriched rat skeletal muscle mitochondrial protein isolates, we generated a catalogue of over 200 carbonylated proteins by virtue of their quantitative enrichment compared to the control sample. The catalogue contains many mitochondrial localized proteins shown to be susceptible to carbonyl modification for the first time, including numerous transmembrane proteins involved in oxidative phosphorylation. Other oxidative modifications (e.g. nitrosylation, hydroxylation) were also identified on many of the carbonylated proteins, providing further evidence of the susceptibility of these proteins to oxidative damage. The results also demonstrate the utility of PQD operation on the LTQ instrument for quantitative analysis of iTRAQ reagent-labeled peptide mixtures, as well as the quantitative reproducibility of the avidin-affinity enrichment method.