Anal. Chem. 2006, 78, 493-500
Performance of a Linear Ion Trap-Orbitrap Hybrid for Peptide Analysis John R. Yates,*,† Daniel Cociorva,† Lujian Liao,† and Vlad Zabrouskov‡
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, and ThermoElectron, Inc., 433 River Oaks Parkway, San Jose, California 95134
Proteomic analysis of digested complex protein mixtures has become a useful strategy to identify proteins involved in biological processes. We have evaluated the use of a new mass spectrometer that combines a linear ion trap and an Orbitrap to create a hybrid tandem mass spectrometer. A digested submandibular/sublingual saliva sample was used for the analysis. We find the instrument is capable of mass resolution in excess of 40 000 and mass measurement accuracies of less than 2 ppm for the analysis of complex peptide mixtures. Such high mass accuracy allowed the elimination of virtually any false positive peptide identifications, suggesting that peptides that do not match the specificity of the protease used in the digestion of the sample should not automatically be considered as false positives. Tandem mass spectra from the linear ion trap and from the Orbitrap have very similar ion abundance ratios. We conclude this instrument will be well suited for shotgun proteomic types of analyses. Analysis of protein mixtures has become a key strategy for the study of biological processes. Two approaches are used to analyze proteins using mass spectrometry. Analysis of intact proteins through molecular weight measurement and subsequent fragmentation using methods such as ECD, SORI, and IRMPD is referred to as “top down” proteomics.1-6 When extensive fragmentation is observed, sufficient sequence coverage can be obtained to identify structural features such as posttranslational modifications. When proteins are chemically or enzymatically cleaved to analyze as peptides, the approach is referred to as “bottom up” analysis.7,8 A strength of the bottom up approach is * To whom correspondence should be addressed. Tel: 858-784-8862. Fax: 858-784-8883. E-mail:
[email protected]. † The Scripps Research Institute. ‡ ThermoElectron, Inc. (1) Kelleher, N. L.; Taylor, S. V.; Grannis, D.; Kinsland, C.; Chiu, H. J.; Begley, T. P.; McLafferty, F. W. Protein Sci. 1998, 7, 1796-1801. (2) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563-573. (3) Horn, D. M.; Ge, Y.; McLafferty, F. W. Anal. Chem. 2000, 72, 4778-4784. (4) Ge, Y.; Lawhorn, B. G.; ElNaggar, M.; Strauss, E.; Park, J. H.; Begley, T. P.; McLafferty, F. W. J. Am. Chem. Soc. 2002, 124, 672-678. (5) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (6) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994, 66, 28012808. (7) Johnson, L.; Mollah, S.; Garcia, B. A.; Muratore, T. L.; Shabanowitz, J.; Hunt, D. F.; Jacobsen, S. E. Nucleic Acids Res. 2004, 32, 6511-6518. 10.1021/ac0514624 CCC: $33.50 Published on Web 12/01/2005
© 2006 American Chemical Society
the ability to “shotgun” analyze complex protein mixtures, that is, to proteolytically digest a protein mixture en masse and analyze the contents of the mixture.9 The bottom up approach when used as a shotgun strategy has successfully identified proteins in complexes, organelles, whole cells, and tissues.10-17 Additionally, information about modification sites in proteins can also be obtained but requires strategies that yield high sequence coverage.18,19 The ability to successfully perform shotgun proteomics is dependent on the quality of chromatography separations, high sensitivity, flexibility, and robustness of mass analyzers for MS and MS/MS, and database search algorithms. Improvements in the overall strategy result from technological advances in any of these areas. One important component to the analysis of complex peptide mixtures is the tandem mass spectrometer. Several forms of tandem mass spectrometers have been used for bottom up analysis including triple quadrupoles, 3-D ion traps, linear ion traps, and TOF-TOFs. A “hybrid” mass spectrometer combines features of two different types of mass spectrometer. Hybrid tandem mass spectrometers combine different types of mass analyzers to enhance the capability or performance of the instrument and include devices such as the Q-TOF, Q-ion trap, (8) Syka, J. E.; Marto, J. A.; Bai, D. L.; Horning, S.; Senko, M. W.; Schwartz, J. C.; Ueberheide, B.; Garcia, B.; Busby, S.; Muratore, T.; Shabanowitz, J.; Hunt, D. F. J. Proteome Res. 2004, 3, 621-626. (9) Eng, J.; McCormack, A.; Yates, J. J. Am. Soc. Mass Spectrom. 1994, 5, 976989. (10) Conaway, J. W.; Florens, L.; Sato, S.; Tomomori-Sato, C.; Parmely, T. J.; Yao, T.; Swanson, S. K.; Banks, C. A.; Washburn, M. P.; Conaway, R. C. FEBS Lett. 2005, 579, 904-908. (11) Sickmann, A.; Reinders, J.; Wagner, Y.; Joppich, C.; Zahedi, R.; Meyer, H. E.; Schonfisch, B.; Perschil, I.; Chacinska, A.; Guiard, B.; Rehling, P.; Pfanner, N.; Meisinger, C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13207-13212. (12) Beausoleil, S. A.; Jedrychowski, M.; Schwartz, D.; Elias, J. E.; Villen, J.; Li, J.; Cohn, M. A.; Cantley, L. C.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12130-12135. (13) Yates, J. R., 3rd; Gilchrist, A.; Howell, K. E.; Bergeron, J. J. Nat. Rev. Mol. Cell. Biol. 2005, 6, 702-714. (14) McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R. Anal. Chem. 1997, 69, 767-776. (15) Wu, C. C.; MacCoss, M. J.; Mardones, G.; Finnigan, C.; Mogelsvang, S.; Yates, J. R., 3rd; Howell, K. E. Mol. Biol. Cell 2004, 15, 2907-2919. (16) Washburn, M. P.; Wolters, D.; Yates, J. R., 3rd. Nat. Biotechnol. 2001, 19, 242-247. (17) Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Yates, J. R. Nat. Biotechnol. 2003, 21, 532-538. (18) MacCoss, M. J.; McDonald, W. H.; Saraf, A.; Sadygov, R.; Clark, J. M.; Tasto, J. J.; Gould, K. L.; Wolters, D.; Washburn, M.; Weiss, A.; Clark, J. I.; Yates, J. R., 3rd. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7900-7905. (19) Cheeseman, I. M.; Anderson, S.; Jwa, M.; Green, E. M.; Kang, J.; Yates, J. R., 3rd; Chan, C. S.; Drubin, D. G.; Barnes, G. Cell 2002, 111, 163-172.
Analytical Chemistry, Vol. 78, No. 2, January 15, 2006 493
Figure 1. Schematic for the LTQ-Orbitrap used in these studies. Ions are transmitted from the linear ion trap to the C-trap and then into the Orbitrap.
and linear ion trap-FT-ion cyclotron resonance (LTQ-FTICR). A recent hybrid, the LTQ-FT, combines the tandem mass spectrometry capabilities of the linear ion trap with the high-resolution/ mass accuracy capability of the FTICR. A particular advantage to the ion trap technology is the ability to control the number of ions transferred to the ICR cell minimizing the impact of space charging.8 A new hybrid tandem mass spectrometer of similar design to the LTQ-FT has been developed that combines the linear ion trap and a new type of mass analyzer called the Orbitrap. The proof of principle of the Orbitrap was first described by Makarov.20 Briefly, the Orbitrap consists of an inner (central) and an outer electrode that are used to trap ions in a quadrologarithmic electrostatic potential. Ions revolve about the central electrode and oscillate harmonically along its axis (the z-direction) with a frequency characteristic of their m/z values. Image current signal of these oscillations is converted to a frequency using Fourier transform in a manner similar to the method used in FTICR. The prototype Orbitrap mass analyzer has demonstrated high resolving power, mass accuracy, and high space charge capacity with pulsed ion sources. Injection of electrosprayed ions into the Orbitrap was first described by Hardman and Makarov.21 Recently a linear ion trap was interfaced to the Orbitrap to combine the tandem mass spectrometry capability of the ion trap mass spectrometer with the high resolution and mass accuracy capability of the Orbitrap.22 We evaluated the high scan rate, resolution, mass accuracy, MS/MS sensitivity of the LTQ-Orbitrap for shotgun proteomics analysis of complex peptide mixtures. MATERIALS AND METHODS Collection of Parotid and Submandibular/Sublingual Saliva. Saliva was collected using a protocol that has been used by the Center for Oral Biology at the University of Rochester. Parotid saliva was collected using a Lashley cup that has been described previously,23 and submandibular/sublingual saliva was collected using a custom device, fabricated from silicone rubber (20) Makarov, A. Anal. Chem. 2000, 72, 1156-1162. (21) Hardman, M.; Makarov, A. A. Anal. Chem. 2003, 75, 1699-1705. (22) Horning, S.; Makarov, A.; Denisov, E.; Wieghaus, A.; Malek, R.; Lange, O.; Senko, M. San Antonio, TX, June 5-9, 2005. (23) Gillece-Castro, B. L.; Prakobphol, A.; Burlingame, A. L.; Leffler, H.; Fisher, S. J. J. Biol. Chem. 1991, 266, 17358-17368.
494
Analytical Chemistry, Vol. 78, No. 2, January 15, 2006
impression material. The two glandular saliva secretions were collected from a single donor under an IRB-approved protocol. Saliva was provided by Professor James Melvin of the University of Rochester. Saliva Processing and Digestion. Unless specified, all chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO). The saliva samples were dialyzed with a molecular weight cutoff membrane (5000 MW) in the absence of protease inhibitors and then lyophilized. Five hundred micrograms each of the two anatomically distinct saliva samples were dissolved by 500 µL of Tris-urea buffer (100 mM Tris-HCl, pH 8.5, 8 M urea). The solutions were then precipitated by adding cold trichloroacetate (TCA) to a final concentration of 30%. After isolating the protein precipitate by centrifugation (14000g, 15 min), the pellets were washed twice with cold acetone, and subsequently dried on a SpeedVac. The protein pellets were again resuspended by 200 µL of Tris-urea buffer, reduced by 25 mM tris(2-carboxyethyl)phosphine hydrochloride (Pierce Chemical Co, Rockford, Il), and cysteine alkylated by 30 mM iodoacetamide. After processing, the samples were first digested by endoproteinase LysC (Roche, Mannheim, Germany) (enzyme-to-substrate ratio of ∼1:200) at 37 °C overnight. Sample solutions were diluted to a final urea concentration of 2 M and digested with a common specific enzyme that cleaves C-terminally at Lys and Arg (Roche) (enzyme-tosubstrate ratio of ∼1:50) at 37 °C overnight. The enzyme reaction was terminated by adding 90% formic acid to a final concentration of 3-5%. Description of the LTQ-Orbitrap. The LTQ-Orbitrap mass spectrometer, depicted in Figure 1 is a hybrid system combining a Finnigan LTQ mass spectrometer and an Orbitrap mass analyzer. In operation, the ions from electrospray ion source are first admitted via rf-only multipoles into the linear trap of LTQ.24 A short prescan is used to determine the ion current within mass range of interest and therefore allows storing a user-defined number of ions (automatic gain control, AGC25). Stored ions are then released from the linear trap and transferred via an rf-only octapole into a curved, C-shaped rf-only quadrupole (C-trap). After (24) Schwartz, J. C.; Senko, M. W.; Syka, J. E. J. Am. Soc. Mass Spectrom. 2002, 13, 659-669. (25) Schwartz, J. C.; Zhou, X. G.; Bier, M. E. U.S. Patent 5,572,022, 1996.
losing energy in collisions with the nitrogen bath gas in the C-trap, ions are trapped in it and form a thin thread along its curved axis. Rapid ramping of voltages on electrodes of C-trap ejects ions orthogonally toward the center of curvature of the C-trap. This presents a significant advance compared to the axial ejection described by Hardman and Makarov21 and Hu et al.26 because it provides fast and uniform extraction for large numbers of stored ions. After leaving the C-trap and passing through appropriately curved ion optics, the ions are accelerated to high kinetic energies and converge into tight clouds which are then able to pass through a small entrance aperture and enter the Orbitrap tangentially. On their way from C-trap, ions pass through three stages of differential pumping until they reach the ultrahigh vacuum compartment of the Orbitrap sustained at ∼2 × 10-10 mbar. The short length of the ion optics reduces deleterious time-of-flight effects and thus minimizes differences in mass spectra between LTQ and Orbitrap mass analyzers. Ions are captured in the Orbitrap by rapidly increasing the electric field, and detection of image current from coherent ion packets takes place after voltages have stabilized at ∼3.5 kV.20 Signals from each of the Orbitrap outer electrodes are amplified by a differential amplifier and transformed into a frequency spectrum by fast Fourier transformation. These frequencies relate to axial oscillations of ions along the Orbitrap, which are independent of the energy and spatial spread of the ions. Axial oscillations are initiated by injecting ions at an offset relative to the equator of the Orbitrap, so no additional excitation stage is required. The frequency spectrum is converted into a mass spectrum and processed with Xcalibur software. Because of electrostatic trapping in the Orbitrap, the frequency of the axial oscillations is inversely proportional to the square root of m/z, while cyclotron frequency in FTICR is inversely proportional to m/z. As a result, the resolving power of the Orbitrap mass analyzer diminishes as a square root of m/z, i.e., slower than in FTICR. Resolving power (m/∆m50% at m/z 400 of the Orbitrap could be switched in discrete steps from the maximum of 100 000 (1.9-s acquisition) to 60 000 (1-s acquisition) and then down to 30 000, 15 000, and 7 500 (the latter corresponding to ∼0.25-0.3 s acquisition). The mass range of both the LTQ and Orbitrap is 4000 Da. Mass calibration coefficients were determined for different AGC target values and interpolated for intermediate values. No intensity-dependent corrections of m/z were made for data processing. All the data were acquired using external calibration with a mixture of caffeine, MRFA peptide, and Ultramark 1600 dissolved in in 50:50 v/v water/acetonitrile solution. LC/MS/MS. The peptide mixtures were separated on-line with a Surveyor LC (Thermo Electron, San Jose, CA) using a 100 × 0.15 mm C18 column (Microtech Scientific, Orange, CA) at a flow rate of 1 µL/min using an 80-min 10-80% acetonitrile/water gradient. Both solvents contained 0.1% formic acid. The electrospray voltage of 1.7 kV versus the inlet of the mass spectrometer was used. Peptides were analyzed using a linear trap/Orbitrap (LTQOrbitrap) hybrid mass spectrometer (Thermo Electron Corp., (26) Hu, Q.; Noll, R. J.; Li, H.; Makarov, A.; Hardman, M.; Graham Cooks, R. J. Mass Spectrom. 2005, 40, 430-443.
Bremen, Germany). Ion transmission into the linear trap and further to the Orbitrap was automatically optimized for maximum ion signal for m/z 150-2000 using AGC. The number of accumulated ions for the precursor ion scan on the linear ion trap (LT), Orbitrap, MSn linear ion trap, and MSn Orbitrap were 3 × 104, 5 × 105, 5 × 103, and 2 × 104, respectively. The resolving power of the Orbitrap mass analyzer was set at 60 000 for the precursor ion scans (m/∆m50% at m/z 400) and 7500 for the MSn (m/∆m50% at m/z 400). The flexibility of the LTQ-Orbitrap platform allows the use of the Orbitrap and linear ion trap mass analyzers independently or simultaneously, depending on experimental requirements. In data-dependent LC/MS2 experiments dynamic exclusion was used with two repeat counts, 30-s repeat duration, and 90-s exclusion duration. All spectra were acquired using a single microscan lasting 0.3-1 s. For MS/MS, precursor ions were activated using 25% normalized collision energy at the default activation q of 0.25. Database Searching. Tandem mass spectra were extracted from raw files, and a binary classifier27spreviously trained on a manually validated data setswas used to remove the low-quality MS/MS spectra. The remaining spectra were searched against a human protein database containing 49 078 protein sequences downloaded as FASTA-formatted sequences from EBI-IPI (database version 3.04, released on March 7, 2005, http://www.ebi.ac.uk/IPI). To calculate confidence levels and false positive rates, a decoy database containing the reverse sequences of the 49 078 proteins was appended to the human database,28 and the SEQUEST algorithm9 was used to find the best matching sequences from the combined database. SEQUEST searches were done on an Intel Xeon 80-processor cluster running under the Linux operating system. The peptide mass search tolerance was set to 3 Da. No differential modifications were considered, and the mass of the amino acid cysteine was statically modified by +57.021 46 Da, due to carboxyamidomethylation of the sample. Spectral matches were retained with XCorr and ∆Cn values that produced a false positive rate of 5%, which was derived from the frequency of matches to the decoy reverse database. Retained spectral matches were filtered and reassigned to proteins using DTASelect.29 RESULTS AND DISCUSSION For shotgun proteomic experiments, the performance of the tandem mass spectrometer is an important component of the process. We sought to determine the performance characteristics of the LTQ-Orbitrap for the analysis of a complex mixture of peptides. The configuration of the LTQ-Orbitrap instrument is depicted in Figure 1. A key feature of the instrument is use of a LTQ in the first stage of mass analysis. In this stage, low-resolution mass spectra can be acquired and tandem mass spectrometry performed on selected ions. Tandem mass spectrometry can be performed at a rate of 10 000 precursor ions dissociated per hour. An additional feature is the use of automatic gain control to limit the number of ions admitted into the ion trap. In the LTQ-Orbitrap, (27) Bern, M.; Goldberg, D.; McDonald, W. H.; Yates, J. R., 3rd. Bioinformatics 2004, 20 (Suppl 1), I49-I54. (28) Peng, J.; Elias, J. E.; Thoreen, C. C.; Licklider, L. J.; Gygi, S. P. J. Proteome Res. 2003, 2, 43-50. (29) Tabb, D. L.; McDonald, H. W.; Yates, J. R., III. J. Proteome Res. 2002, 1, 21-36.
Analytical Chemistry, Vol. 78, No. 2, January 15, 2006
495
Figure 2. Based peak LC/MS/MS ion chromatogram for separation of the digested submandibular/sublingual saliva. A 100-min reversedphase gradient was performed.
hybrid ions are ejected out the back of the trap, transmitted to the C-trap, and then injected into the Orbitrap. Controlling the number of ions transmitted to the C-trap and then injected into the Orbitrap helps minimize space charge effects, which can deteriorate mass resolution and accuracy through nonideal behavior of the ions in the Orbitrap electrostatic fields. Ions in the Orbitrap revolve about the spindle electrode at some frequency and move up and down the spindle in the z-direction reflective of their m/z ratios. By recording the time domain signal of the z-direction frequency of motion, a Fourier transform can convert the signal to the frequency domain and m/z values can be calculated. Measurement of frequency domain signals results in improved resolution and mass accuracy. We determined the performance of the instrument for the LC analysis of a complex peptide mixture. A submandibular-sublingual saliva sample was digested and separated by 1-D LC. Ions were electrosprayed directly into the ion trap using a nanoelectrospray ionization interface. An experimental method was used that obtained a scan of the 400-2000 m/z range in the Orbitrap and then three LTQ MS/MS spectra and one spectrum where the MS/MS fragment ions are scanned into the Orbitrap. Thus, during the first scan a single highresolution (60 000 m/∆m50% at m/z 400) scan is acquired parallel to three low-resolution MS/MS scans and followed by a single high-resolution MS/MS (7500 m/∆m50% at m/z 400) scan acquired during the next 0.3 s from the first of the precursor ions used to acquire the low-resolution MS/MS scans. Thus, the duty cycle time for this five-scan experiment including four MS/MS scans is ∼1.3 s. The MS/MS data were used to search the human sequence database, and the identified sequences were used to evaluate the performance of the Orbitrap. Database searches were conducted using a mass tolerance of 3 amu and no enzyme cleavage specificity. A wider mass tolerance is used for the search since it is not uncommon for the data-dependent process to select the 13C-containing isotope ion especially in larger m/z values. The LTQ-Orbitrap automatically attempts to identify the monoisotopic peak; when this is not possible, as in the case for peptides with low S/N, the MS/MS scan is triggered on the most abundant ion 496
Analytical Chemistry, Vol. 78, No. 2, January 15, 2006
instead. For larger peptides, the most abundant peak belongs to 13C isotopomer; hence, its m/z is written in place of the monoisotopic m/z in such a case. Accurate identification of the peptide is still possible since the ion selection window for CID is 4 Da wide, and thus, the MS/MS spectrum is the same as if the monoisotopic ion was selected. When this happens, the m/z value of the 13C isotope ion is recorded in the MS/MS file and thus a molecular mass that is 1 Da larger is used for the search. We do not yet have an automated method to detect the scan events where the 13C-containing ion is selected as the precursor ion in product ion scans. Over the course of the LC/MS/MS analysis, the Orbitrap was set to acquire data at 60 000 resolution (at m/z 400) and scanned a mass range of 400-2000. The complexity of the scan at 51.4 min is shown in Figure 3A. The mass resolution of ions in three different regions of the mass range is shown in Figure 3B-D. Figure 3A shows the low m/z end of the mass spectrum, and the monoisotopic ion at m/z 539.2710 has a resolution of 56 256. Figure 3B shows the middle range of the same m/z scan, and the monoisotopic ion at m/z 739.401 73 has a resolution of 47 025. Figure 3C shows a higher m/z range of the same m/z scan and the monoisotopic ion at m/z 981.501 22 has a resolution of 39 367. Clearly mass resolution decreases as a function of m/z. Mass resolution is observed to decrease with increasing mass by 30% over the range measured. After searching acquired tandem mass spectra through the human sequence database, the measured molecular weights of identified peptides were compared to their predicted values. A representative example is shown in Figure 4 for the peptide AHFSISNSAEDPFIAIHAESKL. The monoisotopic ion was measured at 739.401 73, which translated to a molecular weight of 2383.181 72. The predicted molecular mass for this peptide is 2383.180 66, which deviates from the observed molecular weight by 1.066 millimass units or 1.3 ppm. Table 1 shows a collection of peptides identified from the analysis of the digested saliva protein mixture. This list is representative of the mass accuracies that were achieved in the analysis of peptides exhibiting cleavage at Lys or Arg. The mean of the mass accuracy measurement
Figure 4. Tandem mass spectrometry performed in the LTQ on the parent at m/z 795.401 73 and identified as having the peptide sequence AHFSISNSAEDPFIAIHAESKL (spectra not shown). The measured molecular weight deviates from the predicted value by 1.3 ppm.
Figure 3. (A) Precursor ion mass spectrum obtained at 51.4 min by the Orbitrap showing the complexity of the peptide mixture at this point in the separation. The Orbitrap instrument was set to record mass spectra at a resolution of 60 000 at m/z 400. The labeled m/z values are expanded to show resolution and peak shape in (B-D).
between observed and predicted molecular weights was 1.5 ppm with a standard deviation of 0.96 ppm. It has been suggested in the literature that peptide matches not meeting protease specificity in a database search must represent false positive identifications because in a specific proteolytic digest such peptides should not exist.30,31 The saliva sample was digested using the method of Washburn et al. that employs a two-step process employing Endoproteinase LysC in 8 M urea as the first step followed by specific Lys and Arg digestion
in 2 M urea.16 Despite the use of two proteases known to be highly specific for Arg/Lys and Lys, the presence of partial cleavage specificity would not be surprising. Saliva is known to contain endogenous proteolytic activity and thus, other proteases are in competition with the added protease for substrates.32 The human genome contains a total of 553 genes annotated as proteases or protease homologues, and thus, the presence of more proteases in saliva than those already identified is a possibility.33 Additionally, proteases such as trypsin are well known to possess other proteolytic activities.34 We searched the tandem mass spectra of peptides using no-enzyme specificity and identified peptides with partial Arg/Lys cleavage specificity with high match scores. Six peptides show chymotryptic cleavage specificity at the N-terminus of the peptides, although it is possible these peptides were partially cleaved by one of the endogenous proteases of saliva. Based on the measured molecular weights of these peptides and their predicted molecular weights, these identifications are believed to be correct. Table 2 shows peptides with partial Arg/Lys specificity as well as their mass deviations from expected values identified from the LC/MS/MS analysis. All have mass accuracies less then 2 ppm. The measurements have a mean value of 1.2 ppm and a standard variation of 0.52 ppm. Given the accuracy of the measured molecular weights of these partially cleaved peptides, it is clear they are not false positive identifications. It is also unlikely these peptides are degradation products as the saliva sample was dialyzed, TCA precipitated, and then lyophilized. These data also suggest that partially cleaved peptides should not a priori be dismissed as a false positive identification. In this hybrid instrument, precursor ions are dissociated in the linear ion trap. Fragment ions can be transferred to the Orbitrap and m/z values recorded at high mass accuracy and high resolution. In these experiments, the resolving power was set at 7500 (m/∆m50% at m/z 400) to record the fragment ions transferred from the LTQ to the Orbitrap. Panels A and B in Figure 5 show (30) Keller, A.; Nesvizhskii, A. I.; Kolker, E.; Aebersold, R. Anal. Chem. 2002, 74, 5383-5392. (31) Olsen, J. V.; Ong, S. E.; Mann, M. Mol. Cell Proteomics 2004, 3, 608-614. (32) Kennedy, S.; Davis, C.; Abrams, W. R.; Billings, P. C.; Nagashunmugam, T.; Friedman, H.; Malamud, D. J. Dent. Res. 1998, 77, 1515-1519. (33) Puente, X. S.; Sanchez, L. M.; Overall, C. M.; Lopez-Otin, C. Nat. Rev. Genet. 2003, 4, 544-558. (34) Inagami, T.; Sturtevant, J. M. J. Biol. Chem. 1960, 235, 1019-1023.
Analytical Chemistry, Vol. 78, No. 2, January 15, 2006
497
Table 1. Peptides Identified in the Digested Saliva Samplea type
charge
sequence
XCorr
∆Cn
theor mass
expl mass
ppm
FT LTQ LTQ LTQ LTQ FT LTQ LTQ FT LTQ LTQ LTQ
+2 +2 +2 +2 +2 +2 +3 +3 +2 +2 +2 +3
K.TGSGDIENYNDATQVR.D K.IAEYMNHLIDIGVAGFR.I K.PFIYQEVIDLGGEPIK.S K.NWGEGWGFMPSDR.A R.DFPAVPYSGWDFNDGK.C R.GHGAGGASILTFWDAR.L K.AHFSISNSAEDPFIAIHAESK.L R.NVVDGQPFTNWYDNGSNQVAFGR.G R.TSIVHLFEWR.W R.ALVFVDNHDNQR.G K.YFSTTEDYDHEITGLR.V K.LDGQISSAYPSQEGQVLVGIYGQYQLLGIK.S
4.81 5.40 5.87 3.84 4.12 5.55 5.15 5.10 3.20 3.55 5.30 6.91
0.44 0.42 0.35 0.37 0.43 0.49 0.38 0.46 0.25 0.32 0.44 0.42
870.3951 959.9958 909.4878 769.8277 907.9046 808.4024 757.7062 862.3983 644.3458 714.3549 973.9419 1075.5661
870.3969 959.9965 909.4898 769.8291 907.9053 808.4038 757.7065 862.3985 644.3458 714.3567 973.9445 1075.5688
2.0 0.8 2.2 1.8 0.7 1.7 0.4 0.3 0.0 2.5 2.6 2.5
a These peptides are cleaved after Arg/Lys residues. Tandem mass spectra used for the identification were obtained on the ion trap (LTQ) or the Orbitrap (FT). SEQUEST scores are shown for each identification. Identified sequences are shown in the form Y.XXXXX.Z, where Y represents the amino acid on the N-terminal side of the cleavage site and Z represents the amino acid on the C-terminal side of the cleavage site.
Table 2. Peptides Identified in the Digested Saliva Samplea MS/MS type
charge
sequence
XCorr
∆Cn
theor mass
expl mass
ppm
2 2 2 2 2 2 2 2 2 2 3 3
N.AVSAGTSSTCGSYFNPGSR.D M.NHLIDIGVAGFR.I F.TNWYDNGSNQVAFGR.G D.FPAVPYSGWDFNDGK.C L.VFVDNHDNQR.G D.VNDWVGPPNDNGVTK.E D.WVGPPNDNGVTK.E D.GQPFTNWYDNGSNQVAFGR.G F.SLTLQTGLPAGTYCDVISGDK.I F.SISNSAEDPFIAIHAESK.L H.FSISNSAEDPFIAIHAESK.L D.GQPFTNWYDNGSNQVAFGR.G
5.50 2.94 4.34 4.75 3.00 3.91 3.06 5.40 4.69 5.60 3.72 4.67
0.46 0.20 0.36 0.35 0.36 0.31 0.22 0.45 0.46 0.44 0.40 0.33
953.4234 656.3620 864.8898 850.3911 622.2943 806.3917 642.3225 1079.4904 1098.5462 958.4734 688.3408 719.9960
953.4248 656.3626 864.8903 850.3921 622.2955 806.3931 642.3236 1079.4921 1098.5473 958.4739 688.3411 719.9968
1.5 0.9 0.5 1.1 1.9 1.8 1.6 1.6 1.0 0.5 0.4 1.0
LTQ LTQ LTQ LTQ FT LTQ LTQ LTQ LTQ LTQ LTQ LTQ
a These peptides are cleaved after Arg/Lys residues. Tandem mass spectra used for the identification were obtained on the ion trap (LTQ) or the Orbitrap (FTMS). SEQUEST scores are shown for the identified peptides. Identified sequences are shown in the form Y.XXXXX.Z, where Y represents the amino acid on the N-terminal side of the cleavage site and Z represents the amino acid on the C-terminal side of the cleavage site.
Table 3. Predicted and Observed Fragment Ions m/z Values at a Mass Resolution of 7500 for the Peptide TSIVHLFEWR b-ions SEQ
no.
T S I V H L F E W R
1 2 3 4 5 6 7 8 9 10
observed
302.16925 401.23843 538.29785 651.38409 798.45178 927.49139 1113.57275
y-ions predicted 102.05496 189.08699 302.17105 401.23946 538.29837 651.38243 798.45084 927.49343 1113.57274 1269.67385
the tandem mass spectrum of the peptide TSIVHLFEWR with fragment ions recorded in the ion trap and Orbitrap, respectively. In the transfer process, fragment ion abundances are maintained between the linear ion trap and Orbitrap. Table 3 shows the measured and predicted m/z values for fragment ions observed in tandem mass spectrum acquired in the Orbitrap. High resolutions and mass accuracies are noted to improve the accuracy of de novo interpretation of tandem mass spectra of peptides, particularly for de novo interpretation using computer algorithms. In general, the appearances of tandem mass spectra in the linear 498 Analytical Chemistry, Vol. 78, No. 2, January 15, 2006
∆
6.0 2.6 1.0 -2.5 -1.2 -2.2 0.0
observed
1099.60132 986.5191 887.4505 750.3918 637.30908 490.24146 361.1980
predicted 1287.68441 1186.63673 1099.60470 986.520637 887.452227 750.393317 637.309257 490.240847 361.198257 175.118947
∆
3.1 1.6 1.9 2.0 0.3 -1.3 0.7
no. 10 9 8 7 6 5 4 3 2 1
ion trap and Orbitrap are quite similar. We calculated a similarity index based on the LibQuest algorithm as a ratio of the scores for LTQ v. FTMS/LTQ v. LTQ.35 An average value of 6 was observed in this comparison for a set of 706 tandem mass spectra out of a possible 10. Scores are not higher because of the resolution difference of the two instruments and the lower level of noise in the FTMS tandem mass spectra. A histogram for the analysis of the spectra is shown in Figure S1 (Supporting (35) Yates, J. R., 3rd; Morgan, S. F.; Gatlin, C. L.; Griffin, P. R.; Eng, J. K. Anal. Chem. 1998, 70, 3557-3565.
Figure 5. (A) Low-resolution tandem mass spectrum obtained for the peptide TSIVHLFEWR (eluting at 49.82 min, Figure 2) in the linear ion trap mass spectrometer. (B) Mass spectrum of the same MS/MS fragments acquired in the Orbitrap mass spectrometer. The predicted versus observed m/z values for the fragment ions are shown in Table 3.
Information). A value greater than 2.5 is considered to be a positive match. Based on this quantitative comparison, we conclude that moving fragment ions between the linear ion trap and the Orbitrap results in minimal distortion in the appearance of the tandem mass spectrum. The analysis of phosphopeptides is of particular interest to understand the regulation of enzyme activity. We identified a phosphopeptide using the LTQ-Orbitrap. The measured molecular weight of the peptide is 3462.7232 and the molecular weight is predicted to be 3462.7009 which is within 6.4 ppm of the measured value (Figure S2, Supporting Information). Additionally no β elimination of phosphate is observed in the Orbitrap mass
spectrum, indicating the ion is transferred from the linear ion trap without loss of phosphate. This peptide was verified as a phosphopeptide by its tandem mass spectrum. The annotated tandem mass spectrum of the phosphopeptide is shown in Supporting Information, Figure S3A. The ions marked in this spectrum are singly charged fragment ions. Because of the large size of the peptide and limited mass range of the ion trap, only a limited amount of sequence is covered. When doubly charged ions are assigned (Supporting Information, Figure S3B), it is apparent many of the higher mass fragment ions are present as doubly charged ions. This information provides much more sequence coverage by fragment ions allowing the identification of the Analytical Chemistry, Vol. 78, No. 2, January 15, 2006
499
modification site. The increased mass accuracy of the precursor ion helps to identify this peptide as a phosphopeptide even though it is a large, multiply charged ion. CONCLUSIONS The analysis of complex peptide mixtures using the LTQOrbitrap clearly results in the acquisition of high-quality peptide data. The acquisition of peptide mass spectra with a mass accuracy of 1-3 ppm and resolutions of 40-60 000 at 1 scan/s are possible. High mass accuracy measurement helps to simplify peptide and protein identifications in proteomic experiments and eliminate nonobvious false positives. Lipton et al. have also shown that accurate mass measurements in the absence of tandem mass spectra can be used to identify peptides from microorganisms,36 and clearly, the mass accuracy of the LTQ-Orbitrap reaches the level required to adopt an “accurate mass tag” approach. Features of this hybrid mass spectrometer important for bottom up proteomics are high scan rates, high resolving power, and high mass accuracy measurements of precursor and product ions and the high fidelity between LTQ and Orbitrap mass spectra. This (36) Lipton, M. S.; Pasa-Tolic, L.; Anderson, G. A.; Anderson, D. J.; Auberry, D. L.; Battista, J. R.; Daly, M. J.; Fredrickson, J.; Hixson, K. K.; Kostandarithes, H.; Masselon, C.; Markillie, L. M.; Moore, R. J.; Romine, M. F.; Shen, Y.; Stritmatter, E.; Tolic, N.; Udseth, H. R.; Venkateswaran, A.; Wong, K. K.; Zhao, R.; Smith, R. D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049-11054.
500
Analytical Chemistry, Vol. 78, No. 2, January 15, 2006
should allow the use of algorithms for the de novo analysis of the ion trap tandem mass spectra with much more accuracy. The combination of an accurate molecular weight with high resolution and accurate fragment ion measurements will result in better and more confident sequence analysis. ACKNOWLEDGMENT Funding for this research was derived in part from NIH grants U01 DE16267, P41 RR11823-09, and R01 MH067880. We thank Dr. Iain Mylchreest of ThermoElectron for access to the LTQOrbitrap. NOTE ADDED AFTER ASAP PUBLICATION The paper was posted December 1, 2005. Minor changes were made to the title and text. The article was reposted on December 6, 2005. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review August 15, 2005. Accepted November 2, 2005. AC0514624
