Anal. Chem. 2009, 81, 3440–3447
Improved Electrospray Ionization Efficiency Compensates for Diminished Chromatographic Resolution and Enables Proteomics Analysis of Tyrosine Signaling in Embryonic Stem Cells Scott B. Ficarro,†,‡,§ Yi Zhang,†,‡ Yu Lu,†,§ Ahmadali R. Moghimi,† Manor Askenazi,†,‡,§,| Elzbieta Hyatt,⊥ Eric D. Smith,†,‡ Leah Boyer,# Thorsten M. Schlaeger,# C. John Luckey,⊥ and Jarrod A. Marto*,†,‡,§ Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, 44 Binney Street, Smith 1158A, Boston, Massachusetts 02115-6084, Department of Biological Chemistry and Molecular Pharmacology, and Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, Stem Cell Program and Division of Hematology/Oncology, Children’s Hospital Boston, Boston, Massachusetts 02115, and Department of Biological Chemistry, The Hebrew University of Jerusalem, Israel Characterization of signaling pathways in embryonic stem cells is a prerequisite for future application of these cells to treat human disease and other disorders. Identification of tyrosine signaling cascades is of particular interest but is complicated by the relatively low levels of tyrosine phosphorylation in embryonic stem cells. These hurdles correlate with the primary limitations of mass spectrometrybased proteomics; namely, poor detection limit and dynamic range. To overcome these obstacles, we fabricated miniaturized LC-electrospray assemblies that provided ∼15-fold improvement in LC-MS performance. Significantly, our characterization data demonstrate that electrospray ionization efficiency compensates for diminished chromatographic performance at effluent flow rates below Van Deemter minima. Use of these assemblies facilitated quantitative proteomics-based analysis of tyrosine signaling cascades in embryonic stem cells. Our results suggest that a renewed focus on miniaturized LC coupled to ultralow flow electrospray will provide a viable path for proteomic analysis of primary cells and rare posttranslational modifications. To date, pluripotent cells have been isolated from the inner cell mass of blastocysts (ESC), 1 cultured postimplantation epiblasts, 2,3 and reprogrammed differentiated fibroblasts (iPS). 4-8 Each of these pluripotent cell populations can in principle undergo * To whom correspondence should be addressed. E-mail: jarrod_marto@ dfci.harvard.edu. Phone: (617) 632-3150. Fax: (617) 582-7737. † Department of Cancer Biology, Dana-Farber Cancer Institute. ‡ Blais Proteomics Center, Dana-Farber Cancer Institute. § Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School. | The Hebrew University of Jerusalem. ⊥ Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School. # Children’s Hospital Boston. (1) Boiani, M.; Scholer, H. R. Nat. Rev. Mol. Cell Biol. 2005, 6, 872–884. (2) Brons, I. G.; Smithers, L. E.; Trotter, M. W.; Rugg-Gunn, P.; Sun, B.; Chuva de Sousa Lopes, S. M.; Howlett, S. K.; Clarkson, A.; Ahrlund-Richter, L.; Pedersen, R. A.; Vallier, L. Nature 2007, 448, 191–195.
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programmed differentiation to generate nearly any cell type. Thus for human pluripotent cells, there is growing excitement that these cells may form the basis of new therapies to fight disease. The majority of large-scale data for ESC are based on genomewide transcriptional profiles and interrogation of transcription factor binding sites. 9 By comparison a relatively small number of protein signaling pathways have been functionally linked to selfrenewal and differentiation in ESC. For example, leukemia inhibitory factor (LIF) maintains self-renewal in murine ESC (mESC) via signaling through the receptor g130, resulting in STAT3 phosphorylation and activation,10 while self-renewal in human ESC (hESC) is dependent upon basic fibroblast growth factor (bFGF) signaling.11,12 Similarly, activity of selected Srcfamily kinases,13,14 glycogen synthethase kinase 3 beta (GSK), and phospho-inositol 3- kinase (PI3K) have been implicated in both self-renewal and differentiation.15-17 Collectively, these results (3) Tesar, P. J.; Chenoweth, J. G.; Brook, F. A.; Davies, T. J.; Evans, E. P.; Mack, D. L.; Gardner, R. L.; McKay, R. D. G. Nature 2007, 448, 196–199. (4) Okita, K.; Ichisaka, T.; Yamanaka, S. Nature 2007, 448, 313–317. (5) Takahashi, K.; Tanabe, K.; Ohnuki, M.; Narita, M.; Ichisaka, T.; Tomoda, K.; Yamanaka, S. Cell 2007, 131, 861–872. (6) Park, I. H.; Zhao, R.; West, J. A.; Yabuuchi, A.; Huo, H.; Ince, T. A.; Lerou, P. H.; Lensch, M. W.; Daley, G. Q. Nature 2008, 451, 141–146. (7) Wernig, M.; Meissner, A.; Foreman, R.; Brambrink, T.; Manching, K.; Hochedlinger, K.; Bernstein, B. E.; Jaenisch, R. Nature 2007, 448, 318– 324. (8) Maherali, N.; Sridharan, R.; Xie, W.; Utikal, J.; Eminli, S.; Arnold, K.; Stadtfeld, M.; Yachechko, R.; Tchieu, J.; Jaenisch, R.; Plath, K.; Hochedlinger, K. Cell Stem Cell 2007, 1, 55–70. (9) Boyer, L. A.; Mathur, D.; Jaenisch, R. Curr. Opin. Genet. Dev. 2006, 16, 455–462. (10) Kristensen, D. M.; Kalisz, M.; Nielsen, J. H. Apmis 2005, 113, 756–772. (11) Daheron, L.; Opitz, S. L.; Zaehres, H.; Lensch, M. W.; Andrews, P. W.; Itskovitz-Eldor, J.; Daley, G. Q. Stem Cells 2004, 22, 770–778. (12) Humphrey, R. K.; Beattie, G. M.; Lopez, A. D.; Bucay, N.; King, C. C.; Firpo, M. T.; Rose-John, S.; Hayek, A. Stem Cells 2004, 22, 522–530. (13) Anneren, C.; Cowan, C. A.; Melton, D. A. J. Biol. Chem. 2004, 279, 31590– 31598. (14) Meyn, M. A., 3rd; Schreiner, S. J.; Dumitrescu, T. P.; Nau, G. J.; Smithgall, T. E. Mol. Pharmacol. 2005, 68, 1320–1330. (15) Sato, N.; Meijer, L.; Skaltsounis, L.; Greengard, P.; Brivanlou, A. H. Nat. Med. 2004, 10, 55–63. (16) Watanabe, S.; Umehara, H.; Murayama, K.; Okabe, M.; Kimura, T.; Nakano, T. Oncogene 2006, 25, 2697–2707. 10.1021/ac802720e CCC: $40.75 2009 American Chemical Society Published on Web 03/30/2009
illustrate that multiple and interdependent signaling cascades lay the molecular groundwork for irreversible cell fate decisions. Hence, methods that facilitate analysis of signaling events in ESC will provide valuable information in conjunction with studies aimed at directed differentiation for therapeutic or tissue engineering applications. Mass spectrometry-based proteomics analysis of pluripotent embryonic stem cells (ESC) has focused primarily on global protein profiling, 18,19 with identification of only a few specific sites of phosphorylation. 20 The latter result is not unexpected given that detection of phosphorylation events in primary or other karyotypically normal cells may be confounded by the rarity of the cells themselves, intrinsically low signaling activity, or some combination thereof. Moreover, and independent of absolute signaling levels, it is typically the case that biological samples enriched by cell type, protein class, or specific post-translational modification, contain a concentration dynamic range that exceeds the analytical capabilities of current LC-MS instruments. In principle, improvements to the sample introduction interface for mass spectrometry can provide gains in detection limit and dynamic range. Recent efforts to advance proteomics sample introduction for LC-MS focused on either the use of small columns and particles for so-called fast separations21-26 or platforms with robotically controlled nozzle arrays to introduce LC effluent;27 arguably these approaches emphasize reproducibility and robustness over optimum chromatographic and electrospray performance. Interestingly, the respective advantages of small inner diameter (I.D.), capillary-based liquid chromatography for improved separation performance28-31 and very low flow electrospray for improved ionization efficiency32-36 were independently described in multiple publications over a decade ago. However, the difficulty inherent to fabrication of the small and robust emitter tips required to support stable electrospray at increasingly low (17) Paling, N. R.; Wheadon, H.; Bone, H. K.; Welham, M. J. J. Biol. Chem. 2004, 279, 48063–48070. (18) Ma, L.; Sun, B.; Hood, L.; Tian, Q. Clin. Chim. Acta 2007, 378, 24–32. (19) Graumann, J.; Hubner, N. C.; Kim, J. B.; Ko, K.; Moser, M.; Kumar, C.; Cox, J.; Scholer, H.; Mann, M. Mol. Cell. Proteomics 2008, 7, 672–683. (20) Puente, L. G.; Borris, D. J.; Carriere, J. F.; Kelly, J. F.; Megeney, L. A. Mol. Cell. Proteomics 2006, 5, 57–67. (21) Castro-Perez, J.; Plumb, R.; Granger, J. H.; Beattie, I.; Joncour, K.; Wright, A. Rapid Commun. Mass Spectrom. 2005, 19, 843–848. (22) Lippert, J. A.; Xin, B.; Wu, N.; Lee, M. L. J. Microcolumn Sep. 1999, 11, 631–643. (23) Issaeva, T.; Kourganov, A.; Unger, K. J. Chromatogr. A 1999, 846, 13–23. (24) MacNair, J. E.; Opiteck, G. J.; Jorgenson, J. W.; Moseley, M. A. Rapid Commun. Mass Spectrom. 1997, 11, 1279–1285. (25) MacNair, J. E.; Patel, K. D.; Jorgenson, J. W. Anal. Chem. 1999, 71, 700– 708. (26) Tolley, L.; Jorgenson, J. W.; Moseley, M. A. Anal. Chem. 2001, 73, 2985– 2991. (27) Corso, T. N.; VanPelt, C. K.; Li, J.; Ptak, C.; Huang, X. Anal. Chem. 2006, 78, 2209–2219. (28) Hsieh, S.; Jorgenson, J. W. Anal. Chem. 1996, 68, 1212–1217. (29) Karlsson, K. E.; Novotny, M. Anal. Chem. 1988, 60, 1662–1665. (30) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 1128–1135. (31) Novotny, M. Anal. Chem. 1988, 60, 500A–510A. (32) Gale, D. C.; Smith, R. D. Rapid. Commun. Mass Spectrom. 1993, 7, 1017– 1021. (33) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605– 613. (34) Valaskovic, G. A.; Kelleher, N. L.; Little, D. P.; Aaserud, D. J.; McLafferty, F. W. Anal. Chem. 1995, 67, 3802–3805. (35) Wilm, M.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167– 180. (36) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1–8.
effluent flow rates34,35,37 has exacerbated efforts to decipher the relative performance compromise between otherwise divergent trends in chromatographic resolution and electrospray ionization efficiencies expected in low flow regimes. Anecdotal evidence suggests that researchers struggle to find a suitable convergence between key metrics that will facilitate exploration of small-scale, low flow LC-MS assemblies. Despite these obstacles we anticipated that fabrication of miniaturized LC-ESI assemblies would facilitate exploration of experimental parameters heretofore inaccessible and ultimately provide a viable path for proteomics-based analysis of low-level tyrosine signaling in ESC or other primary cells. EXPERIMENTAL SECTION Construction of Fused Silica Analytical Columns with Integrated Emitter Tips. Analytical columns (AC) were constructed from 360 µm O.D. × 50 µm I.D. and 360 µm O.D. × 25 µm I.D. fused silica capillary tubing (O.D., outer diameter; I.D., inner diameter). Figure 1a depicts the major features of these columns, with images that illustrate discrete steps during fabrication (b-f). Silicate-based frits were cast in situ based on the following protocol. A 2.5 cm section of polyimide was removed approximately 3 cm from one end of the fused silica tubing. A silicate solution was allowed to migrate via capillary action to the midpoint of the exposed window (Figure 1b). Next, polymerization was induced using a soldering iron as described above, with care taken to form frits of 1-2 mm in length (Figure 1c). After ejection of excess silicate solution the frits were reheated with the soldering iron at 400 °C for several seconds. Reversed phase beds were packed as described above, with 5 µm diameter beads used for the 50 µm I.D. AC and 3 µm diameter beads used in the 25 µm I.D. AC. For both column sizes bed lengths of 12 cm were attained in 10 min or less at pressures of 500-1500 psi. Next, the columns were dried with pressurized helium for 5 min (Figure 1d). Panel (e) shows a cross-section image of a fritted 25 µm I.D. capillary, taken at the frit midpoint. Finally, an integrated emitter tip of 0.75-1.5 µm diameter (Figure 1f) was formed 2-4 mm beyond the frit using a laser-based pipet puller (P-2000, Sutter Instruments, Novato, CA). A Nikon I.C.-66 microscope (Micro Optics, Fresh Meadows, NY) was used at 1000 × or 1500 × magnification for visual confirmation of tip diameter. Prior to first use the end of the column (opposite the tip) was cleaved to within
