Zeptomole-Sensitivity Electrospray Ionization -Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Proteins Mikhail E. Belov, Mikhail V. Gorshkov, ² Harold R. Udseth, Gordon A. Anderson, and Richard D. Smith* Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352 Methods are being developed for ultrasensitive protein characterization based upon electrospray ionization (ESI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The sensitivity of a FTICR mass spectrometer equipped with an ESI source depends on the overall ion transmission, which combines the prob- ability of ionization, transmission efficiency, and ion trapping in the FTICR cell. Our developments imple- mented in a 3.5 tesla FTICR mass spectrometer include introduction and optimization of a newly designed elec- trodynamic ion funnel in the ESI interface, improving the ion beam characteristics in a quadrupole-electrostatic ion guide interface, and modification of the electrostatic ion guide. These developments provide a detection limit of approximately 3 0 zmol (∼1 8 0 0 0 molecules) for proteins with molecular weights ranging from 8 to 2 0 kDa. Electrospray ionization mass spectrometry ( ESI-M S) has become widely used for the study of biopolymers. 1-3 Sensitivity is often a major issue for many biological applications of ESI-MS (e.g., in proteomics, protein analysis from a single cell, and sample- limited applications in general). Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in conjunction with electrospray ionization provides powerful analytical capabilities for ultrasensitive protein characterization. 4-6 Improvements to the design of ESI sources have given rise to detection limits in the femtomole to attomole range. 7-9 Further increases in the sensitiv- ity of FTICR mass spectrometers depend significantly upon increasing the overall ion transmission from solution to the FTICR cell as well as the efficiency of trapping the ions in the cell. While electrospray ionization at atmospheric pressure can be very efficient for dilute samples flowing delivered to the electrospray emitter at low flow rates, the reduction or elimination of losses during ion transport from the atmospheric pressure region of the ESI source to the second vacuum stage at a pressure of few hundred milliTorr, where transmission becomes more efficient, has been a challenge. The recently developed “electrodynamic ion funnel” has demonstrated a substantial improvement in the ion transport efficiency through the first vacuum stage (1-5 Torr) of a mass spectrometer. 10-12 An rf field applied to the funnel electrodes creates an effective potential which confines the ion beam radially in the presence of a buffer gas, while a dc axial gradient moves the ions toward the exit electrode. The ion funnel focuses ions entering from atmospheric pressure more efficiently through a conductance-limiting orifice. This results in an effective matching to the acceptance area of a rf-only multipole ion guide, thus minimizing ion losses during ion transfer to the next lower- pressure region of the spectrometer. In this work we report on the performance of a 3.5 tesla (T) FTICR mass spectrometer equipped with an ESI source and demonstrate high-sensitivity performance for proteins and pep- tides. The ESI source incorporates a newly designed ion funnel whose initial performance has recently been described. 14 Com- pared with the previously reported ion funnel design, 10-12 the new ion funnel has improved significantly the transmission for the total ion current, broadened m/ z transmission range, and reduced collisional activation in the interface. These improvements have enabled detection limits in low zeptomolar range. Permanent address: Institute of Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia 117829 * To whom correspondence should be addressed (1) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984 , 88, 4451. (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science ( Washington, D.C.) 1989 , 246, 64-71. (3) Smith, R. D.; Loo, J. A.; Edmonds, C. G. Anal. Chem. 1991 , 63, 2488. (4) Valaskovic, G. A.; Kelleher, N. L.; Little, D. P.; Aaserud, D. J.; McLaferty, F. W. Anal. Chem. 1995 , 67, 3802-3805. (5) Hofstadler, S. A.; Severs, J. C.; Smith, R. D.; Swanek, P. D.; Ewing, A. G. Rapid Commun. Mass Spectrom. 1996 , 10, 919-922. (6) Emmett, M. R.; White, F. M.; Hendrickson, C. L.; Shi, S. D.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1998 , 9, 333-340. (7) Wahl, J. H.; Goodlet, D. R.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1992 , 64, 3194-3196. (8) Andren, P. E.; Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994 , 5, 867-869. (9) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science (Washington, D.C.) 1996 , 273, 1199-1201. (10) Shaffer, S. A.; Tang, K.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1997 , 11, 1813-1817. (11) Shaffer, S. A.; Prior, D. C.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1998 , 70, 4111-4119. (12) Shaffer, S. A.; Tolmachev, A. V.; Prior, D. C.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1999 , 71, 2957-2964. (13) Dodonov, A.; Kozlovsky, V.; Loboda, A.; Raznikov, V.; Sulimenkov, I.; Tolmachev, A.; Kraft, A.; Wollnik, H. Rapid Commun. Mass Spectrom. 1997 , 11, 1649-1656. (14) Belov, M. E.; Gorshkov, M. V.; Udseth, H. R.; Anderson, G. A.; Tolmachev, A. V.; Prior, D. C.; Harkewicz, R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2000 , 11, 19-23. Anal. Chem. 2000, 72, 2271-2279 10.1021/ac991360b CCC: $19.00 © 2000 American Chemical Society Analytical Chemistry, Vol. 72, No. 10, May 15, 2000 2271 Published on Web 04/12/2000