Robust and Sensitive iTRAQ Quantification on an LTQ Orbitrap Mass Spectrometer

Marcus Bantscheff,Markus Boesche,Dirk Eberhard,Toby Matthieson,Gavain Sweetman,Bernhard Kuster

doi:10.1074/mcp.m800029-mcp200

Abstract

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.

Highlights

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
Pulsed Q Dissociation (PQD) Optimization—isobaric tags for relative and absolute quantification (iTRAQ)-labeled Fibrinopeptide A (FibA) was used to optimize instrument parameters for PQD. iTRAQ 114 and iTRAQ 117-labeled FibA was mixed at a ratio of 1:0.8 to yield a total concentration of 330 fmol/␮l and continuously infused into the mass spectrometer at 250 nl/min using a syringe pump
At collision energy of 29%, by far the most abundant ion in the PQD spectrum is intact doubly charged FibA (m/z 841.3). iTRAQ reporter ions were detected at ϳ20% relative intensity, and similar intensities were observed for other fragments (Fig. 1D)

Summary

EXPERIMENTAL PROCEDURES

Sample Preparation—Carboxymethylated BSA was purchased from Michrom Bioresources Inc. (Auburn, AL), and Fibrinopeptide A was purchased from Bachem AG (Bubendorf, CH). Reduced and carbamidomethylated kinobead eluates were concentrated on 4 –12% NuPAGE gels (Invitrogen) by running sample ϳ1 cm into the gel to remove reagents incompatible with tryptic digestion and iTRAQ labeling. For Q-Tof Ultima data, 0.4 Da mass tolerance was allowed for intact peptide and fragment ions (for a plot of the distribution of measured precursor mass deviation see supplemental Fig. S1). Peptide and Protein Quantification—Centroided iTRAQ reporter ion signals were computed by the XCalibur software operating the mass spectrometer and extracted from MS data files using in-house developed software. For the BSA dilution series, we required iTRAQ reporter ions to be detected under all conditions applied and reporter ion areas to be above the electronic noise level (200 for PQD spectra and 2000 for HCD spectra)

RESULTS

DISCUSSION

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