Anal. Chem. 2005, 77, 2176-2186
Improvement in Peptide Detection for Proteomics Analyses Using NanoLC-MS and High-Field Asymmetry Waveform Ion Mobility Mass Spectrometry Karine Venne,† Eric Bonneil,†,‡ Kevin Eng,† and Pierre Thibault*,†,‡
Caprion Pharmaceuticals, Montre´ al, Canada H4S 2C8, and Institute for Research in Immunology and Cancer, Universite´ de Montre´ al, Montreal, Canada H3C 3J7
Sensitive and selective detection of multiply charged peptide ions from complex tryptic digests was achieved using high-field asymmetric waveform ion mobility spectrometry (FAIMS) combined with nanoscale liquid chromatography-mass spectrometry (nanoLC-FAIMS-MS). The combination of FAIMS provided a marked advantage over conventional nanoLC-MS experiments by reducing the extent of chemical noise associated with singly charged ions and enhancing the overall population of detectable tryptic peptides. Such advantages were evidenced by a 6-12-fold improvement in signal-to-noise ratio measurements for a wide range of multiply charged peptide ions. An increase of 20% in the number of detected peptides compared to conventional nanoelectrospray was achieved by transmitting ions of different mobilities at high electric field vs low field while simultaneously recording each ion population in separate mass spectrometry acquisition channels. This method provided excellent reproducibility across replicate nanoLC-FAIMS-MS runs with more than 90% of all detected peptide ions showing less than 30% variation in intensity. The application of this technique in the context of proteomics research is demonstrated for the identification of trace-level proteins showing differential expression in U937 monocyte cell extracts following incubation with phorbol ester. The past decade has witnessed an unparallel expansion of mass spectrometry into cell biology and protein chemistry, and the technological developments that led to this growth paved the way to numerous large-scale proteomics projects. Proteomics analyses entail significant analytical challenges far exceeding the current capabilities of modern mass spectrometry instrumentation. Indeed, the scale and magnitude of biological complexity represented by the variability in protein content and cellular distribution, the dynamic range in protein expression (> 106-fold), the multitude of protein modifications, and the limited sample availability typify the challenges facing proteomics investigations1. * Corresponding author. E-mail:
[email protected]. Phone: (514) 343-6910. Fax: (514) 343-7586. † Caprion Pharmaceuticals. ‡ University de Montre´al. (1) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207.
2176 Analytical Chemistry, Vol. 77, No. 7, April 1, 2005
Efforts to decipher the inherent complexity of cellular proteomes have focused on the development of analytical approaches providing sample separation via targeted enrichment, organelle fractionation, or both2,3 enhanced peak capacity and dynamic range in protein detection.4,5 To this end, nanoscale capillary liquid chromatography with on-line mass spectrometry detection (nanoLC-MS) continues to play a central role in any mass spectrometry-based proteomics program. Protein identification is typically performed on gel-isolated proteins previously separated by one- or two-dimensional gel electrophoresis. Proteolytic peptides resulting from in-gel tryptic digestion are then separated by reversed-phase chromatography and sequenced via collisional activation of selected precursor ions in a tandem mass spectrometer (MS-MS).1 Alternatively, other proteomics approaches avoid gels altogether and involves in-solution protein digestion followed by two-dimensional liquid chromatography (strong cation exchange and reversed-phase C18 chromatography) prior to mass spectrometry detection.6,7 Current tandem mass spectrometry instruments typically provide reliable peptide sequencing for low-femtomole levels of protein digests. While successful protein identification relies on the correlation of observed fragment ions with those predicted from nonredundant protein databases,8-11 unambiguous assignment also depends on the abundance and complexity of the primary ions selected for sequencing.12,13 Indeed, the sequencing (2) Jung, E.; Heller, M.; Sanchez, J. C.; Hochstrasser, D. F. Electrophoresis 2000, 21, 3369-3377. (3) Brunet, S.; Thibault, P.; Gagnon, E.; Kearney, P.; Bergeron, J. J. M.; Desjardins, M. Trends Cell Biol. 2003, 13, 629-638. (4) Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriguez, N.; Smith, R. D. Anal. Chem. 2002, 74, 4235-4249. (5) Shen, Y.; Moore, R. J.; Zhao, R.; Blonder, J.; Auberry, D. L.; Masselon, C.; Pasa-Tolic, L.; Hixsen, K.; Auberry, K. J.; Smith, R. D. Anal. Chem. 2003, 75, 3264-3273. (6) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682. (7) Wolters, D. A.; Washburn, M. P.; Yates, J. R., III. Anal. Chem. 2001, 73, 5683-5690. (8) Eng, J. K.; McCormack, A. L.; Yates, J. R. J. Am. Soc., Mass Spectrom. 1994, 5, 976-989. (9) Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 4390-4399. (10) Clauser, K. R.; Baker, P. R.; Burlingame A. L. Anal. Chem. 1999, 71, 28712882. (11) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567. (12) Bern, M.; Goldberg, D.; McDonald, W. H.; Yates, J. R. Bioinformatics 2004, 20, i49-i54. 10.1021/ac048410j CCC: $30.25
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of tryptic peptides becomes increasingly more challenging at subfemtomole levels due to the higher proportion of transient chemical interference ions associated with the ionization of the mobile-phase constituents, column bleed, siloxane adduct ions,14 or charge competition and ionization suppression effects.15 Clearly, enhancement in the sensitivity of MS-MS for peptide sequencing relies on technological developments enabling the selection of target ions while minimizing the contribution of undesired precursor ions of similar m/z values. To this end, ion selection based on gas-phase mobility characteristics of peptide ions offers an interesting approach to improve the sensitivity and selectivity of mass spectrometry. Previous reports have described the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) to reduce chemical noise16 and enhance peptide detection.17 FAIMS is a gasphase ion separation technique based on compound-dependent differences in ion mobility at high-field, Kh (∼104 V/cm) relative to low-field, K (
