title: Discovery- and target-based protein quantification using iTRAQ and pulsed Q collision induced dissociation (PQD).
abstract: Pulsed Q collision-induced dissociation (PQD) was developed in part to facilitate detection of low-mass reporter ions using labeling reagents (e.g. iTRAQ) on LTQ platforms. It has generally been recognized that the scan speed and sensitivity of an LTQ are superior than those of an Orbitrap using the higher-energy collisional dissociation (HCD). However, the use of PQD in quantitative proteomics is limited, primarily due to the meager reproducibility of reporter ion ratios. Optimizations of PQD for iTRAQ quantification using LTQ have been reported, but a universally applicable strategy for quantifying the less abundant proteins has not been fully established. Adjustments of the AGC target, μscan, or scan speed offer only incremental improvements in reproducibility. From our experience, however, satisfactory coefficients of variation (CVs) of reporter ion ratios were difficult to achieve using the discovery-based approach. As an alternative, we implemented a target-based approach that obviates data dependency to allow repetitive data acquisitions across chromatographic peaks. Such a strategy generates enough data points for more reliable quantification. Using cAMP treatment in S49 cell lysates and this target-based approach, we were able to validate differentially expressed proteins, which were initially identified as potential candidates using the discovery-based PQD. The target-based strategy also yielded results comparable to those obtained from HCD in an Orbitrap. Our findings should aid LTQ users who desire to explore iTRAQ quantitative proteomics but have limited access to the more costly Orbitrap or other instruments.
authors.affiliation: Metabolism Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: Wells W
authors.initials: WW
authors.lastname: Wu
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wang
authors.forename: Paul A
authors.initials: PA
authors.lastname: Insel
authors.forename: Cheng-Te
authors.initials: CT
authors.lastname: Hsiao
authors.forename: Sige
authors.initials: S
authors.lastname: Zou
authors.forename: Bronwen
authors.initials: B
authors.lastname: Martin
authors.forename: Stuart
authors.initials: S
authors.lastname: Maudsley
authors.forename: Rong-Fong
authors.initials: RF
authors.lastname: Shen
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D005591:Chemical Fractionation / Q000379:methods*; D055808:Drug Discovery / Q000379:methods; D019220:High-Energy Shock Waves; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding / Q000528:radiation effects; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D054928:Validation Studies as Topic
chemical_list: D011506:Proteins