Anal. Chem. 2006, 78, 1337-1344
Quantitative Comparison of Proteomic Data Quality between a 2D and 3D Quadrupole Ion Trap Adele R. Blackler,† Aaron A. Klammer,‡ Michael J. MacCoss,‡ and Christine C. Wu*,†
Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, and Department of Genome Sciences, University of Washington, Seattle, Washington 98198
A 2D ion trap has a greater ion trapping efficiency, greater ion capacity before observing space-charging effects, and a faster ion ejection rate than a traditional 3D ion trap mass spectrometer. These hardware improvements should result in a significant increase in protein identifications from complex mixtures analyzed using shotgun proteomics. In this study, we compare the quality and quantity of peptide identifications using data-dependent acquisition of tandem mass spectra of peptides between two commercially available ion trap mass spectrometers (an LTQ and an LCQ XP Max). We demonstrate that the increased trapping efficiency, increased ion capacity, and faster ion ejection rate of the LTQ results in greater than 5-fold more protein identifications, better identification of low-abundance proteins, and higher confidence protein identifications when compared with a LCQ XP Max. In the past few years, there has been an explosion in technological advances improving the performance of commercially available mass spectrometers. A particularly powerful development has been the two-dimensional (2D) radio frequency (rf) quadrupole ion trap. Multipole-based 2D ion trap mass analyzers have been used extensively as part of hybrid mass spectrometers, including Fourier transform ion cyclotron resonance, time-of-flight, and triple quadruple mass analyzers. However, more recently, the 2D ion trap mass analyzer is being used as a stand-alone mass spectrometer instead of solely as part of a hybrid instrument. One commercially available 2D ion trap mass spectrometer is the ThermoElectron LTQ. This mass spectrometer is based on the standard three-dimensional ion trap, the ThermoElectron LCQ. Unlike the LCQ, which contains two end cap electrodes and a ring electrode, the LTQ is composed of a segmented hyperbolic quadrupole mass analyzer with three distinct axial segments. The quadruple mass analyzer creates a 2D rf field, and discrete dc voltages are applied to the different axial segments to contain the ions axially in the center segment of the mass analyzer. A slit is cut in two of the center rods to facilitate radial ion ejection and detection at two electron multiplier detectors. Unlike alternative 2D ion trap mass spectrometers, many of the scan functions and electronics on the LTQ operate in a fashion similar to the 3D ion * To whom correspondence should be addressed. Phone: 303-724-3351. E-mail:
[email protected]. † University of Colorado Health Sciences Center. ‡ University of Washington. 10.1021/ac051486a CCC: $33.50 Published on Web 01/17/2006
© 2006 American Chemical Society
trapsusing resonance excitation for ion isolation, activation, and mass analysis.1 Although the 2D ion trap operates in a fashion analogous to that of a conventional 3D ion trap, it has several significant performance advantages. First, there is no rf field in the ion injection axis for the ions to overcome before entering the trap. Therefore, the trapping efficiency from an external ion source is improved dramatically. This improved trapping efficiency results in a much faster relative ion injection time and contributes to a more rapid duty cycle. Second, the linear configuration of the mass analyzer results in a larger volume that improves the overall ion capacity before observing space-charging effects. This increased ion capacity is complemented by the radial ejection of ions, as opposed to axial ejection on a 3D ion trap. Radial ejection of ions facilitates the use of two detectors, one on each side of the mass analyzer, resulting in a ∼2× increase in total detection efficiency. Finally, the LTQ has a ∼3× increase in ion ejection rate relative to the LCQ while maintaining the same resolution. These three improvementssincreased trapping efficiency, ion capacity, and ion ejection ratesshould significantly improve the qualitative identification of peptides by automated µLC/MS/MS. To demonstrate these improvements, identical complex peptide mixtures of soluble Escherichia coli proteins were analyzed by datadependent µLC/MS/MS on ThermoElectron LTQ and LCQ XP Max mass spectrometers, and the resulting tandem MS spectra were searched using a normalized version of SEQUEST. The program DTASelect was used to apply thresholds that maximized the number of protein identifications while minimizing the false discovery rate (FDR) for both data sets. The LTQ data set resulted in a 5-fold higher number of protein identifications, including a higher percentage of identifications from low-abundance proteins, and increased spectral count and sequence coverage for identified proteins. METHODS Sample Preparation. E. coli (strain OP50) was cultured in LB media to log phase growth at 37 °C. Bacteria were pelleted and lysed in 50 mM NH4HCO3 pH 8.5 at 1000 psi using a French press. Total lysates were microfuged at 14 000 rpm for 30 min at 4 °C. The resulting supernatant was collected and assayed for protein concentration using the RC DC Protein Assay Kit (BioRad, Hercules, CA). Protein samples were reduced in 5 mM dithio(1) Schwartz, J. C.; Senko, M. W.; Syka, J. E. J. Am. Soc. Mass Spectrom. 2002, 13, 659-669.
Analytical Chemistry, Vol. 78, No. 4, February 15, 2006 1337
theitol for 30 min at 60 °C and alkylated in the dark in 15 mM iodoacetamide for 30 min at 25 °C. The protein sample was then adjusted to 0.1% Rapigest (Waters Corp., Milford, MA) and 2 mM CaCl2 and digested using modified trypsin (Roche, Indianapolis, IN) at a ratio of 1:30 enzyme/protein overnight at 37 °C with shaking. Digestion was terminated by acidification with 200 mM HCl and incubation at 37 °C for 45 min. The sample was microfuged for 4 min at 14 000 rpm, and the supernatant was collected for proteomic analysis. Microcapillary liquid chromatography-tandem mass spectrometry (µLC/MS/MS). Identical samples were analyzed five separate times on both an LCQ XP Max and an LTQ ion trap mass spectrometer. Both instruments had the ThermoElectron Ion Max electrospray source replaced with identical in-house-constructed microspray sources and were interfaced with identical Agilent 1100 binary HPLC and autosampler systems. Each protein digest (10 µg) was loaded from the autosampler onto a fused-silica capillary column (100-µm i.d.) packed with 15 cm of Luna C18 material (Phenomonex) mounted in the microspray source using flow from the HPLC pump. The flow during the loading was split prior to the autosampler from 150 to ∼2 µL/min. After 25 min of loading, the location of the split was changed from upstream of the autosampler to immediately distal the microcapillary column using the divert valve on both mass spectrometers. The restriction of the “running” split was less than the restriction of the “loading” split reducing the flow through the column from ∼2 µL/min to ∼500 nL/min. Mass spectra were acquired using data-dependent acquisition with a single full mass scan followed by three MS/MS scans. Each MS/MS scan acquired was an average of three microscans on the LCQ and two microscans on the LTQ. Data Analysis. MS/MS spectra from each analysis were searched using no enzyme specificity on a 96-node G5 Beowulf cluster against an NCBI E. coli-protein database concatenated to sequences of common contaminants and a shuffled decoy database2 using a normalized implementation of SEQUEST.3 The resulting peptide identifications were assembled into proteins using DTASelect,4 thresholds were adjusted, and the FDR monitored using the decoy database. Default thresholds cutoffs were made using the following parameters: normalized crosscorrelation score for +1, +2, and +3 charge peptides of 0.28, DeltCN value of 0.15, include only fully tryptic peptides, allow loci with a single peptide identification, peptides must have >4 amino acids, 20% of the predicted fragment ions must be accounted for within the spectrum, remove protein identifications that were subsets of others, and remove ambiguous identifications. Using DTASelect, these filters can be achieved using the following command line parameters: -1 0.28-2 0.28-3 0.28 -d .15 -y 2 -p 1 -i 0.2 -o -Smn 4 -a false. The FDR was estimated by dividing the number of protein identifications mapping to proteins from the shuffled decoy database by the number of nonredundant proteins mapping to the unshuffled protein sequences. Microarray Data. Publicly available microarray data were used as a proxy measure of abundance for the proteins identified (2) Finney, G.; Merrihew, G.; Klammer, A.; Frewen, B.; MacCoss, M. J. In 53rd ASMS Conference on Mass Spectrometry; San Antonio, TX, 2005. (3) MacCoss, M. J.; Wu, C. C.; Yates, J. R., 3rd. Anal. Chem. 2002, 74, 55935599. (4) Tabb, D. L.; McDonald, W. H.; Yates, J. R., 3rd. J. Proteome Res. 2002, 1, 21-26.
1338 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006
Table 1. Summary of Data Collected for Five Independent Runs on Both the LTQ and LCQ XP Maxa total spectra MS and MS/MS
no. of MS/MS spectra
no. of protein IDs
FDR
LTQ 29097 ( 1079* 18784 ( 654 566 ( 7* 0.25 ( 0.12 LCQ XP Max 5789 ( 189 4087 ( 127 100 ( 4 0.15 ( 0.16 a
Data are expressed as mean ( standard deviation. *P
