Anal. Chem. 2005, 77, 2406-2414
Statistical Evaluation of Internal and External Mass Calibration Laws Utilized in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry David C. Muddiman*,† and Ann L. Oberg‡
W.M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center and Department of Biochemistry and Molecular Biology, and Division of Biostatistics, Mayo Clinic College of Medicine, Rochester, Minnesota 55905
The statistical evaluation of two common and three new calibration laws utilized in Fourier transform ion cyclotron resonance mass spectrometry are presented. Electrospray ionization was used to prepare a series of mass spectra of ammonium-adducted polypropylene glycol (PPG) with an average molecular weight of 1000 Da. The singly charged PPG-1000 oligomers allowed for the description of a broad range of m/z and abundance values within each mass spectrum. The hexapole accumulation time was varied to afford a range of total ion abundance values of about an order of magnitude. To examine each of the calibration laws, we utilized cross-validation both “withinspectrum” and “between-spectra” for internally and externally calibrated data, respectively. In addition, we used t-statistics to ensure that each calibration coefficient was statistically significant and necessary to accurately describe the variation in the data. In comparison to commonly used calibration laws for internal calibration, our new calibration law based on multiple linear regression offered a 2-fold improvement in mass measurement accuracy (MMA). In comparison to external calibration laws without automatic gain control, our new calibration law using multiple regression improved the MMA by >10fold; this improvement would increase further as the dynamic range of the measurement increases (e.g., a biological system). For both our internal and external calibration laws, the median MMA was less than 1 partper-million. Furthermore, we investigate the number of calibrant ions as well as their required m/z range in order to successfully achieve high MMA. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), first introduced in 1974 by Comisarow and Marshall,1 has undergone significant advances over the past 30 years. The * To whom correspondence should be addressed: David C. Muddiman, Ph.D., Medical Sciences Building 3-115, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905. Phone: 507-284-1997. Fax: 507-284-9261. E-mail:
[email protected]. † W.M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center and Department of Biochemistry and Molecular Biology. ‡ Division of Biostatistics. (1) Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 25, 282-283.
2406 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005
coupling of electrospray ionization2 with FTICR by McLafferty and Hunt3 marked the beginning of an era of tremendous impact and continuing opportunities in biological research. Several recent reviews have elegantly described the extraordinary potential of FTICR-MS in the area of proteomics,4,5 metabolomics,6 glycomics,7 and genomics.8,9 This is attributed to the unparalleled analytical performance of FTICR including high mass measurement accuracy (MMA), dynamic range, resolving power, and sensitivity as well as offering of a diverse range of tandem-MS methods including electron capture dissociation,10,11 sustained off-resonance irradiation,12,13 infrared multiphoton dissociation,13,14 and hybrid instruments;15-17 the first quadrupole-FTICR (QFT) was reported by McIver and co-workers in 1985.18 The accurate determination of the monoisotopic mass of a peptide is an extraordinarily powerful approach to increase the confidence of protein identification,19,20 especially when coupled with other constraints such as enzyme specificity, retention time,21 (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (3) Henry, K. D.; Williams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9075-9078. (4) Bogdanov, B.; Smith, R. Mass Spectrom. Rev., published online Mar 30, 2004. (5) Meng, F.; Forbes, A.; Miller, L.; Kelleher, N. Mass Spectrom. Rev., published online Mar 30, 2004. (6) Brown, S.; Kruppa, G.; Dasseux, J.-L. Mass Spectrom. Rev., published online Mar 30, 2004. (7) Park, Y.; Lebrilla, C. Mass Spectrom. Rev., published online Mar 30, 2004. (8) Null, A. P.; Muddiman, D. C. J. Mass Spectrom. 2001, 36, 589-606. (9) Hofstadler, S.; Sannes-Lowery, K.; Hannis, J. Mass Spectrom. Rev., published online Mar 30, 2004. (10) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc 1998, 120, 3265-3266. (11) Cooper, H.; Håkansson, K.; Marshall, A. Mass Spectrom. Rev., published online Mar 30, 2004. (12) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (13) Laskin, J.; Futrell, J. Mass Spectrom. Rev., published online Mar 30, 2004. (14) Little, D. P.; Speir, J. P.; Senko, M. W.; Oconnor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (15) Wang, Y.; Shi, S. D.-H.; Henrickson, C. L.; Marshall, A. G. Int. J. Mass Spectrom. Ion Proc. 2000, 198, 113-120. (16) Harkewicz, R.; Belov, M. E.; Anderson, G. A.; Pasa-Tolic, L.; Masselon, C. D.; Prior, D. C.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2002, 13, 144-154. (17) 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. (18) McIver, J. R. T.; Hunter, R. L.; Bowers, W. D. Int. J. Mass Spectrom. Ion Proc. 1985, 64, 67-77. 10.1021/ac048258l CCC: $30.25
© 2005 American Chemical Society Published on Web 03/10/2005
presence of cysteine,22 and augmentation with sequence information.23-25 Marshall and co-workers have provided a critical insight regarding using accurate mass measurements for protein identification.26 FTICR mass spectrometry has the ability to define the mass of a molecular species with unparalleled accuracy (∼1 part-permillion). This can be accomplished if the space-charge frequency shifts, introduced by the ions themselves, are taken into account.27,28 McIver and co-workers demonstrated in 1983 that the observed cyclotron frequency was inversely proportional to the number of ions present in the ICR cell.28 Thus, external mass calibration must accurately account for these systematic frequency shifts in order to routinely achieve high MMA. Internal calibration requires the presence of an internal standard in each collected spectrum and is most readily achieved using a dual-ESI source,29-34 although other approaches have been successful.35,36 Internal calibration is attractive because the frequencies of the calibrant ions are exposed to the same ICR cell conditions as the ions under investigation. However, this method is not commercially available and requires a higher level of skill to implement properly, and interference of calibrant with analyte ions could result, especially when dealing with the complex mixtures routinely encountered when studying biological systems. External calibration can be accomplished by two different strategies. Amster and co-workers37 used a calibration curve to correct for the total ion abundance in the ICR cell relative to the external calibration spectrum. This resulted in routinely achieving
