Picoelectrospray Ionization Mass Spectrometry Using Narrow-Bore Chemically Etched Emitters

  • Ioan Marginean
  • Keqi Tang
  • Richard D. Smith
  • Ryan T. Kelly
Research Article


Electrospray ionization mass spectrometry (ESI-MS) at flow rates below ~10 nL/min has been only sporadically explored because of difficulty in reproducibly fabricating emitters that can operate at lower flow rates. Here we demonstrate narrow orifice chemically etched emitters for stable electrospray at flow rates as low as 400 pL/min. Depending on the analyte concentration, we observe two types of MS signal response as a function of flow rate. At low concentrations, an optimum flow rate is observed slightly above 1 nL/min, whereas the signal decreases monotonically with decreasing flow rates at higher concentrations. For example, consumption of 500 zmol of sample yielded signal-to-noise ratios ~10 for some peptides. In spite of lower MS signal, the ion utilization efficiency increases exponentially with decreasing flow rate in all cases. Significant variations in ionization efficiency were observed within this flow rate range for an equimolar mixture of peptide, indicating that ionization efficiency is an analyte-dependent characteristic for the present experimental conditions. Mass-limited samples benefit strongly from the use of low flow rates and avoiding unnecessary sample dilution. These findings have important implications for the analysis of trace biological samples.

Key words

Nanoelectrospray nano-ESI Quantitation Mass-limited analysis 



The authors thank William F. Danielson for writing the syringe pump control software, and Sarah Rausch, Allison Sheen, and Levi Broeske for assistance with data processing. This research was supported by the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL) intramural program and grants from National Institutes of Health: the National Institute of General Medical Sciences (grant 8 P41 GM103493-10) and the National Cancer Institute (1R33CA155252). The EMSL is a national scientific user facility sponsored by US DOE’s Office of Biological and Environmental Research and located at the Pacific Northwest National Laboratory (PNNL) in Richland, WA. PNNL is a multiprogram national laboratory operated by Battelle for the DOE under contract no. DE-AC05-76RLO 1830.


  1. 1.
    Hopfgartner, G., Bean, K., Henion, J., Henry, R.: Ion-spray mass-spectrometric detection for liquid chromatography—a concentration-flow-sensitive or a mass-flow-sensitive device. J. Chromatogr. 647, 51–61 (1993)CrossRefGoogle Scholar
  2. 2.
    Davis, M.T., Stahl, D.C., Hefta, S.A., Lee, T.D.: A microscale electrospray interface for online, capillary liquid-chromatography tandem mass-spectrometry of complex peptide mixtures. Anal. Chem. 67, 4549–4556 (1995)CrossRefGoogle Scholar
  3. 3.
    Ikonomou, M.G., Blades, A.T., Kebarle, P.: Electrospray ion spray—a comparison of mechanisms and performance. Anal. Chem. 63, 1989–1998 (1991)CrossRefGoogle Scholar
  4. 4.
    Kostiainen, R., Bruins, A.P.: Effect of multiple sprayers on dynamic-range and flow-rate limitations in electrospray and ionspray mass-spectrometry. Rapid Commun. Mass Spectrom. 8, 549–558 (1994)CrossRefGoogle Scholar
  5. 5.
    Hopfgartner, G., Wachs, T., Bean, K., Henion, J.: High-flow ion spray liquid-chromatography mass-spectrometry. Anal. Chem. 65, 439–446 (1993)CrossRefGoogle Scholar
  6. 6.
    Wahl, J.H., Goodlett, D.R., Udseth, H.R., Smith, R.D.: Attomole level capillary electrophoresis mass-spectrometric protein-analysis using 5-um-i.d. capillaries. Anal. Chem. 64, 3194–3196 (1992)CrossRefGoogle Scholar
  7. 7.
    Gale, D.C., Smith, R.D.: Small-volume and low flow-rate electrospray-ionization mass-spectrometry of aqueous samples. Rapid Commun. Mass Spectrom. 7, 1017–1021 (1993)CrossRefGoogle Scholar
  8. 8.
    Emmett, M.R., Caprioli, R.M.: Micro-electrospray mass-spectrometry-ultra-high-sensitivity analysis of peptides and proteins. J. Am. Soc. Mass Spectrom. 5, 605–613 (1994)CrossRefGoogle Scholar
  9. 9.
    Wilm, M.S., Mann, M.: Electrospray and Taylor-cone theory, Dole’s beam of macromolecules at last. Int. J. Mass Spectrom. Ion Process. 136, 167–180 (1994)CrossRefGoogle Scholar
  10. 10.
    Bateman, K.P., White, R.L., Thibault, P.: Disposable emitters for on-line capillary zone electrophoresis nanoelectrospray mass spectrometry. Rapid Commun. Mass Spectrom. 11, 307–315 (1997)CrossRefGoogle Scholar
  11. 11.
    Oosterkamp, A.J., Gelpi, E., Abian, J.: Quantitative peptide bioanalysis using column-switching nano liquid chromatography mass spectrometry. J. Mass Spectrom. 33, 976–983 (1998)CrossRefGoogle Scholar
  12. 12.
    Chang, Y.Z., Chen, Y.R., Her, G.R.: Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated tapered fused-silica capillary with a beveled edge. Anal. Chem. 73, 5083–5087 (2001)CrossRefGoogle Scholar
  13. 13.
    Chen, Y.R., Tseng, M.C., Her, G.R.: Design and performance of a low-flow capillary electrophoresis-electrospray-mass spectrometry interface using an emitter with dual beveled edge. Electrophoresis 26, 1376–1382 (2005)CrossRefGoogle Scholar
  14. 14.
    Schmidt, A., Karas, M., Dulcks, T.: Effect of different solution flow rates on analyte ion signals in nano-ESI MS, or: when does ESI turn into nano-ESI? J. Am. Soc. Mass Spectrom. 14, 492–500 (2003)CrossRefGoogle Scholar
  15. 15.
    Tang, X.T., Bruce, J.E., Hill, H.H.: Characterizing electrospray ionization using atmospheric pressure ion mobility spectrometry. Anal. Chem. 78, 7751–7760 (2006)CrossRefGoogle Scholar
  16. 16.
    Marginean, I., Kelly, R.T., Prior, D.C., LaMarche, B.L., Tang, K.Q., Smith, R.D.: Analytical characterization of the electrospray ion source in the nanoflow regime. Anal. Chem. 80, 6573–6579 (2008)CrossRefGoogle Scholar
  17. 17.
    Heemskerk, A.A.M., Busnel, J.M., Schoenmaker, B., Derks, R.J.E., Klychnikov, O., Hensbergen, P.J., Deelder, A.M., Mayboroda, O.A.: Ultra-low flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics. Anal. Chem. 84, 4552–4559 (2012)Google Scholar
  18. 18.
    Geromanos, S., Freckleton, G., Tempst, P.: Tuning of an electrospray ionization source for maximum peptide-ion transmission into a mass spectrometer. Anal. Chem. 72, 777–790 (2000)CrossRefGoogle Scholar
  19. 19.
    Gucek, M., Vreeken, R.J., Verheij, E.R.: Coupling of capillary zone electrophoresis to mass spectrometry (MS and MS/MS) via a nanoelectrospray interface for the characterisation of some beta-agonists. Rapid Commun. Mass Spectrom. 13, 612–619 (1999)CrossRefGoogle Scholar
  20. 20.
    Bendahl, L., Hansen, S.H., Olsen, J.: A new sheathless electrospray interface for coupling of capillary electrophoresis to ion-trap mass spectrometry. Rapid Commun. Mass Spectrom. 16, 2333–2340 (2002)CrossRefGoogle Scholar
  21. 21.
    Ishihama, Y., Katayama, H., Asakawa, N., Oda, Y.: Highly robust stainless steel tips as micro electro spray emitters. Rapid Commun. Mass Spectrom. 16, 913–918 (2002)CrossRefGoogle Scholar
  22. 22.
    Chen, Y.R., Her, G.R.: A simple method for fabrication of silver-coated sheathless electrospray emitters. Rapid Commun. Mass Spectrom. 17, 437–441 (2003)CrossRefGoogle Scholar
  23. 23.
    Tseng, M.C., Chen, Y.R., Her, G.R.: A beveled tip sheath liquid interface for capillary electrophoresis-electrospray ionization-mass spectrometry. Electrophoresis 25, 2084–2089 (2004)CrossRefGoogle Scholar
  24. 24.
    Tang, K.Q., Page, J.S., Smith, R.D.: Charge competition and the linear dynamic range of detection in electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 15, 1416–1423 (2004)CrossRefGoogle Scholar
  25. 25.
    Wilm, M., Mann, M.: Analytical properties of the nanoelectrospray ion source. Anal. Chem. 68, 1–8 (1996)CrossRefGoogle Scholar
  26. 26.
    Valaskovic, G.A., Kelleher, N.L., McLafferty, F.W.: Attomole protein characterization by capillary electrophoresis mass spectrometry. Science 273, 1199–1202 (1996)CrossRefGoogle Scholar
  27. 27.
    Bahr, U., Pfenninger, A., Karas, M., Stahl, B.: High sensitivity analysis of neutral underivatized oligosaccharides by nanoelectrospray mass spectrometry. Anal. Chem. 69, 4530–4535 (1997)CrossRefGoogle Scholar
  28. 28.
    El-Faramawy, A., Siu, K.W.M., Thomson, B.A.: Efficiency of nano-electrospray ionization. J. Am. Soc. Mass Spectrom. 16, 1702–1707 (2005)CrossRefGoogle Scholar
  29. 29.
    Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., Mann, M.: Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469 (1996)CrossRefGoogle Scholar
  30. 30.
    Shen, Y.F., Zhao, R., Berger, S.J., Anderson, G.A., Rodriguez, N., Smith, R.D.: High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics. Anal. Chem. 74, 4235–4249 (2002)CrossRefGoogle Scholar
  31. 31.
    Chang, Y.W., Zhao, C.X., Wu, Z.M., Zhou, J., Zhao, S.M., Lu, X., Xu, G.W.: Chip-based nanoflow high performance liquid chromatography coupled to mass spectrometry for profiling of soybean flavonoids. Electrophoresis 33, 2399–2406 (2012)CrossRefGoogle Scholar
  32. 32.
    Zhou, F., Lu, Y., Ficarro, S.B., Webber, J.T., Marto, J.A.: Nanoflow low pressure high peak capacity single dimension LC-MS/MS platform for high-throughput, in-depth analysis of mammalian proteomes. Anal. Chem. 84, 5133–5139 (2012)CrossRefGoogle Scholar
  33. 33.
    Kelly, R.T., Page, J.S., Luo, Q.Z., Moore, R.J., Orton, D.J., Tang, K.Q., Smith, R.D.: Chemically etched open tubular and monolithic emitters for nanoelectrospray ionization mass spectrometry. Anal. Chem. 78, 7796–7801 (2006)CrossRefGoogle Scholar
  34. 34.
    Marginean, I., Kelly, R.T., Page, J.S., Tang, K.Q., Smith, R.D.: Electrospray characteristic curves: in pursuit of improved performance in the nanoflow regime. Anal. Chem. 79, 8030–8036 (2007)CrossRefGoogle Scholar
  35. 35.
    Valaskovic, G.A., Utley, L., Lee, M.S., Wu, J.T.: Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimation of metabolite levels in drug discovery. Rapid Commun. Mass Spectrom. 20, 1087–1096 (2006)CrossRefGoogle Scholar
  36. 36.
    Kelly, R.T., Page, J.S., Tang, K.Q., Smith, R.D.: Array of chemically etched fused-silica emitters for improving the sensitivity and quantitation of electrospray ionization mass spectrometry. Anal. Chem. 79, 4192–4198 (2007)CrossRefGoogle Scholar
  37. 37.
    Mizuno, H., Tsuyama, N., Harada, T., Masujima, T.: Live single-cell video-mass spectrometry for cellular and subcellular molecular detection and cell classification. J. Mass Spectrom. 43, 1692–1700 (2008)CrossRefGoogle Scholar
  38. 38.
    Mellors, J.S., Jorabchi, K., Smith, L.M., Ramsey, J.M.: Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry. Anal. Chem. 82, 967–973 (2010)CrossRefGoogle Scholar
  39. 39.
    Nemes, P., Knolhoff, A.M., Rubakhin, S.S., Sweedler, J.V.: Metabolic differentiation of neuronal phenotypes by single-cell capillary electrophoresis-electrospray ionization-mass spectrometry. Anal. Chem. 83, 6810–6817 (2011)CrossRefGoogle Scholar
  40. 40.
    Rubakhin, S.S., Romanova, E.V., Nemes, P., Sweedler, J.V.: Profiling metabolites and peptides in single cells. Nat. Methods 8, S20–S29 (2011)CrossRefGoogle Scholar
  41. 41.
    Shrestha, B., Patt, J.M., Vertes, A.: In situ cell-by-cell imaging and analysis of small cell populations by mass spectrometry. Anal. Chem. 83, 2947–2955 (2011)CrossRefGoogle Scholar
  42. 42.
    Stolee, J.A., Shrestha, B., Mengistu, G., Vertes, A.: Observation of subcellular metabolite gradients in single cells by laser ablation electrospray ionization mass spectrometry. Angew. Chem. Int. Ed. 51, 10386–10389 (2012)CrossRefGoogle Scholar
  43. 43.
    Walker, B.N., Stolee, J.A., Vertes, A.: Nanophotonic ionization for ultratrace and single-cell analysis by mass spectrometry. Anal. Chem. 84, 7756–7762 (2012)CrossRefGoogle Scholar
  44. 44.
    Kelly, R.T., Tolmachev, A.V., Page, J.S., Tang, K.Q., Smith, R.D.: The ion funnel: theory, implementations, and applications. Mass Spectrom. Rev. 28, 294–312 (2010)Google Scholar
  45. 45.
    Enke, C.G.: A predictive model for matrix and analyte effects in electrospray ionization of singly-charged ionic analytes. Anal. Chem. 69, 4885–4893 (1997)CrossRefGoogle Scholar
  46. 46.
    Kelly, R.T., Page, J.S., Zhao, R., Qian, W.J., Mottaz, H.M., Tang, K.Q., Smith, R.D.: Capillary-based multi nanoelectrospray emitters: improvements in ion transmission efficiency and implementation with capillary reversed-phase LC-ESI-MS. Anal. Chem. 80, 143–149 (2008)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry (outside the USA) 2013

Authors and Affiliations

  • Ioan Marginean
    • 1
  • Keqi Tang
    • 1
  • Richard D. Smith
    • 1
  • Ryan T. Kelly
    • 2
  1. 1.Biological Sciences DivisionPacific Northwest National LaboratoryRichlandUSA
  2. 2.Environmental Molecular Sciences LaboratoryPacific Northwest National LaboratoryRichlandUSA

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